Histology_Ross

Histology_Ross. Inclusions contain products of metabolic activity of the cell and consist largely of pigment granules, lipid droplets, and glycogen. Inclusions are cytoplasmic or nuclear structures with characteristic staining properties that are formed from the metabolic products of cell. They are considered nonmoving and nonliving components of the cell. Some of them, such as pigment granules, are surrounded by a plasma membrane; others (e.g., lipid droplets or glycogen) instead reside within the cytoplasmic or nuclear matrix.  Lipofuscin is a brownish-gold pigment visible in routine


Histology_Ross

Histology_Ross. H&E preparation. It is easily seen in nondividing cells such as neurons and skeletal and cardiac muscle cells. Lipofuscin accumulates during the years in most eukaryotic cells as a result of cellular senescence (aging); thus, it is often called the “wear-and-tear” pigment. Lipofuscin is a conglom erate of oxidized lipids, phospholipids, metals, and organic molecules that accumulate within the cells as a result of oxidative degradation of mitochondria and lysosomal digestion. Phagocytotic cells such as macrophages may also


Histology_Ross

Histology_Ross. One of the critical components of normal cell division is the precise redistribution of chromosomes and other cell or-ganelles during mitosis. Following replication of chromoso-mal DNA in the S phase of the cell cycle, centrioles undergo a single round of duplication that is closely coor-dinated with cell-cycle progression. During mitosis, centri-oles are responsible for forming the bipolar mitotic spindle, which is essential for equal segregation of chromosomes between daughter cells. Alterations of mechanisms regu-lating centriole duplication may lead to multiplication and abnormalities of centrioles and surrounding centrosomes (MTOCs). Such changes may distort the mitotic spindle (i.e., the presence of multipolar or misoriented spindles) (Fig. F.2.3.1), leading to abnormal sorting of chromosomes during cell divisions. The resulting changes in chromoso-mal number (aneuploidy) may increase the activity of onco-genes or decrease protection from tumor-suppressor genes. These changes


Histology_Ross

Histology_Ross. chromosomes during cell divisions. The resulting changes in chromoso-mal number (aneuploidy) may increase the activity of onco-genes or decrease protection from tumor-suppressor genes. These changes are known to promote malignant cell transformation. Increased numbers of centrioles are frequently observed in tumor cells.


Histology_Ross

Histology_Ross. FOLDER 2.3 Clinical Correlation: Abnormal Duplication of Centrioles and Cancer ababFIGURE F2.3.1 • Multipolar mitotic spindle in a tumor cell. a. Electron micrograph of the invasive breast tumor cell showing abnormal symmetrical tripolar mitotic spindle in the metaphase of cell division. 16,000. b. This drawing composed by color tracings of microtubules (red), mitotic spindle poles (green), and metaphase chromosomes (blue) (obtained from six nonadja-cent serial sections of dividing tumor cell) shows more clearly the organization of this abnormal mitotic spindle. Detailed analy-sis and three-dimensional reconstruction of the spindle revealed that each spindle pole had at least two centrioles and that one spindle pole was composed of two distinct but adjacent foci of microtubules. (Reprinted with permission from Lingle WL. Salisbury JL. Altered centrosome structure is associated with abnormal mitoses in human breast tumors. Am J Path 1999; 155:1941–1951.) contain lipofuscin, which


Histology_Ross

Histology_Ross. with permission from Lingle WL. Salisbury JL. Altered centrosome structure is associated with abnormal mitoses in human breast tumors. Am J Path 1999; 155:1941–1951.) contain lipofuscin, which accumulates from the digestion of bacteria, foreign particles, dead cells, and their own organelles. Recent experiments indicate that lipofuscin accumulation may be an accurate indicator of cellular stress.


Histology_Ross

Histology_Ross.  Hemosiderin is an iron-storage complex found within the cytoplasm of many cells. It is most likely formed by the indigestible residues of hemoglobin, and its presence is related to phagocytosis of red blood cells. Hemosiderin is most easily demonstrated in the spleen, where aged erythrocytes are phagocytosed, but it can also be found in alveolar macrophages in the lung tissue, especially after pulmonary infection accompanied by small hemorrhage into the alveoli. It is visible in light microscopy as a deep brown granule, more or less indistinguishable from lipofuscin. Hemosiderin granules can be differentially stained using histochemical methods for iron detection.


Histology_Ross

Histology_Ross.  Glycogen is a highly branched polymer used as a storage material for glucose. It is not stained in the routine H&E preparation. However, it may be seen in the light microscope with special fixation and staining procedures (such as toluidine blue or the PAS method). Liver and striated muscle cells, which usually contain large amounts of glycogen, may display unstained regions where glycogen is located. Glycogen appears in EM as granules 25 to 30 nm in diameter or as clusters of granules that often occupy significant portions of the cytoplasm (Fig. 2.58).  Lipid inclusions (fat droplets) are usually nutritive inclusions that provide energy for cellular metabolism. The lipid droplets may appear in a cell for a brief time (e.g., in intestinal absorptive cells) or may reside for a long period (e.g., in adipocytes). In adipocytes, lipid inclusions often constitute most of the cytoplasmic vol


Histology_Ross

Histology_Ross. FIGURE 2.57 • Association of the centrosome duplication and primary cilium formation with the cell cycle. After a cell emerges from mitosis, it possesses a single centrosome (MTOC) surrounded by amorphous pericentriolar material. The primary cilium formation frst occurs during G1 phase in which the centrosome migrates toward the cell membrane and initiates the process of ciliogenesis. Necessary structural and transport proteins are acquired and activated to build primary cilium axoneme (9 0) directly on the top of the mature centriole. During the end of G1 phase, as well as in GO, the primary cilium functions as an external receiver antenna sensing and interpreting signals from the extracellular environment. Duplication of centrioles begins near the transition between the G1 and S phases of the cell cycle, and the two centrioles are visible in S phase. During the late G2 phase, centrioles reach their full maturity, whereas the primary cilium is disassembled. This allows centrioles to


Histology_Ross

Histology_Ross. of the cell cycle, and the two centrioles are visible in S phase. During the late G2 phase, centrioles reach their full maturity, whereas the primary cilium is disassembled. This allows centrioles to migrate away from the cell membrane and participate in the mitotic spindle formation. Once cell division is complete, the centrioles can proceed to ciliary reassembly in G1 phase. (Based on Santos N, Reiter JF. Building it up and taking it down: the regulation of vertebrate ciliogenesis. Dev Dyn 2008;237:1972–1981.) ume, compressing the other formed organelles into a thin rim at the margin of the cell. Lipid droplets are usually extracted by the organic solvents used to prepare tissues for both light and electron microscopy. What is seen as a fat droplet in light microscopy is actually a hole in the cytoplasm that represents the site from which the lipid was extracted. In individuals with genetic defects of enzymes involved in lipid metabolism, lipid droplets may accumulate in abnormal


Histology_Ross

Histology_Ross. in the cytoplasm that represents the site from which the lipid was extracted. In individuals with genetic defects of enzymes involved in lipid metabolism, lipid droplets may accumulate in abnormal locations or in abnormal amounts. Such diseases are classified as lipid storage diseases.


Histology_Ross

Histology_Ross.  Crystalline inclusions contained in certain cells are recognized in the light microscope. In humans, such inclusions are found in the Sertoli (sustentacular) and Leydig (interstitial) cells of the testis. With the TEM, crystalline inclusions have been found in many cell types and in virtually all parts of the cell, including the nucleus and most cytoplasmic organelles. Although some of these inclusions contain viral proteins, storage material, or cellular metabolites, the significance of others is not clear. The cytoplasmic matrix is a concentrated aqueous gel consisting of molecules of different sizes and shapes.


Histology_Ross

Histology_Ross. The cytoplasmic matrix (ground substance or cytosol) shows little specific structure by light microscopy or conventional TEM and has traditionally been described as a concentrated aqueous solution containing molecules of different size and shape (e.g., electrolytes, metabolites, RNA, and synthesized proteins). In most cells, it is the largest single compartment. The cytoplasmic matrix is the site of physiologic processes that are fundamental to the cell’s existence (protein synthesis, breakdown of nutrients). Studies with high-voltage EM (HVEM) of 0.25to 0.5-m sections reveal a complex three-dimensional structural network of thin microtrabecular strands and cross-linkers. This network provides a structural substratum on which cytoplasmic reactions occur, such as those involving free ribosomes, and along which regulated and directed cytoplasmic transport and movement of organelles occur.


Histology_Ross

Histology_Ross. FIGURE 2.58 • Electron micrographs of a liver cell with glycogen inclusions. a. Low-magnifcation electron micrograph showing a portion of a hepatocyte with part of the nucleus (N, upper left). Glycogen (G) appears as irregular electron-dense masses. Profles of rough endoplasmic reticulum (rER) and mitochondria (M) are also evident. 10,000. b. This higher-magnifcation EM reveals glycogen (G) as aggregates of small particles. Even the smallest aggregates (arrows) appear to be composed of several smaller glycogen particles. The density of the glycogen is considerably greater than that of the ribosomes (lower left). 52,000. The Cell Nucleus


Histology_Ross

Histology_Ross. The Cell Nucleus OVERVIEW OF THE NUCLEUS / 75 NUCLEAR COMPONENTS / 75 Chromatin / 75 Nucleolus / 79 Nuclear Envelope / 81 Nucleoplasm / 84 CELL RENEWAL / 84 CELL CYCLE / 86 Phases and Checkpoints Within the Cell Cycle / 86 Regulation of the Cell Cycle / 87 Mitosis / 89 Meiosis / 89 CELL DEATH / 93 Apoptosis / 94 Other Forms of Programmed Cell Death / 95 Folder 3.1 Clinical Correlation: Cytogenetic Testing / 80 Folder 3.2 Clinical Correlation: Regulation of Cell Cycle and Cancer Treatment / 81 The nucleus is a membrane-limited compartment that contains the genome (genetic information) in eukaryotic cells. The nucleus contains genetic information, together with the machinery for DNA replication and RNA transcription and processing. The nucleus of a nondividing cell, also called an interphase cell, consists of the following components.


Histology_Ross

Histology_Ross.  Chromatin, is nuclear material organized as euchromatin or heterochromatin. It contains DNA associated with roughly an equal mass of various nuclear proteins (e.g., histones) that are necessary for DNA to function.  The nucleolus (pl., nucleoli) is a small area within the nucleus that contains DNA in the form of transcription-ally active ribosomal RNA (rRNA) genes, RNA, and proteins. The nucleolus is the site of rRNA synthesis and contains regulatory cell-cycle proteins.  The nuclear envelope is the membrane system that surrounds the nucleus of the cell. It consists of an inner and an outer membrane separated by a perinuclear cisternal space and perforated by nuclear pores. The outer membrane of the nuclear envelope is continuous with that of the rough-surfaced endoplasmic reticulum (rER) and is often studded with ribosomes.  The nucleoplasm is nuclear content other than the chromatin and nucleolus.


Histology_Ross

Histology_Ross.  The nucleoplasm is nuclear content other than the chromatin and nucleolus. A simple microscopic evaluation of the nucleus provides a great deal of information about cell well-being. Evaluation of nuclear size, shape, and structure plays an important role in tumor diagnosis. For instance, dying cells have visible nuclear alterations. These include karyolysis, or the disappearance of nuclei due to complete dissolution of DNA by increased activity of DNAase, pyknosis, or condensation of chromatin leading to shrinkage of the nuclei (they appears as dense basophilic masses), and karyorrhexis, or fragmentations of nuclei (these changes usually are proceeded by pyknosis). Chromatin, a complex of DNA and proteins, is responsible for the characteristic basophilia of the nucleus.


Histology_Ross

Histology_Ross. Chromatin, a complex of DNA and proteins, is responsible for the characteristic basophilia of the nucleus. Each eukaryotic cell contains about 6 billion bits of information encoded in DNA structure, which has a total length of about 1.8 meter. The length of the DNA molecule is 100,000 times longer than the nuclear diameter. Therefore, the DNA must be highly folded and tightly packed in the cell nucleus. This is accomplished by the formation of a unique nucleoprotein complex called chromatin. The chromatin complex consists of DNA and structural proteins. Further folding of chromatin, such as that which occurs during mitosis, produces structures called chromosomes. Each human cell contains 46 chromosomes. Chromatin proteins include five basic proteins called histones along with other nonhistone proteins. A unique feature of chromatin packaging is that it permits the transcriptional machinery to access those regions of the chromosomes that are required for gene expression.


Histology_Ross

Histology_Ross. Sequencing human genome was successfully completed in 2003.


Histology_Ross

Histology_Ross. The human genome encompasses the entire length of human DNA that contains the genetic information packaged in all 46 chromosomes. Sequencing of the human genome took about 13 years and was successfully completed in 2003 by the Human Genome Project. The human genome contains a 2.85-billion base pair consensus sequence of nucleotides, which are arranged in about 23,000 protein-coding genes. For years it was thought that genes were usually present in two copies in a genome. However, recent discoveries have revealed that large segments of DNA can vary in numbers of copies. Such copy number variations (CNVs) are widespread in the human genome and most likely lead to genetic imbalances. For instance, genes that were thought to always occur in two copies per genome have sometimes one, three, or more copies. A previous definition of a gene as a segment of DNA involved in producing a polypeptide chain has been recently updated to now read as being a union of genomic sequences encoding a


Histology_Ross

Histology_Ross. or more copies. A previous definition of a gene as a segment of DNA involved in producing a polypeptide chain has been recently updated to now read as being a union of genomic sequences encoding a coherent set of potentially overlapping functional products.


Histology_Ross

Histology_Ross. In general, two forms of chromatin are found in the nucleus: A condensed form called heterochromatin and a dispersed form called euchromatin.


Histology_Ross

Histology_Ross. In most cells, chromatin does not have a homogeneous appearance; rather, clumps of densely staining chromatin are embedded in a more lightly staining background. The densely staining material is highly condensed chromatin called heterochromatin, and the lightly staining material (where most transcribed genes are located) is a dispersed form called euchromatin. It is the phosphate groups of the chromatin DNA that are responsible for the characteristic basophilia of chromatin (page 6). Heterochromatin is disposed in three locations (Fig. 3.1):  Marginal chromatin is found at the periphery of the nu cleus (the structure light microscopists formerly referred to as the nuclear membrane actually consists largely of marginal chromatin).  Karyosomes are discrete bodies of chromatin irregular in size and shape that are found throughout the nucleus.  Nucleolar-associated chromatin is chromatin found in association with the nucleolus.


Histology_Ross

Histology_Ross. Heterochromatin stains with hematoxylin and basic dyes; it is also readily displayed with the Feulgen procedure (a specific histochemical reaction for the deoxyribose of DNA, page 6) and fluorescent vital dyes such as Hoechst dyes and propidium iodide. It is the heterochromatin that accounts for the conspicuous staining of the nucleus in hematoxylin and eosin (H&E) preparations. Euchromatin is not evident in the light microscope. It is present within the nucleoplasm in the “clear” areas between and around the heterochromatin. In routine electron micro-graphs, there is no sharp delineation between euchromatin and heterochromatin; both have a granular, filamentous appearance, but the euchromatin is less tightly packed.


Histology_Ross

Histology_Ross. Euchromatin indicates active chromatin—that is, chromatin that is stretched out so that the genetic information in the DNA can be read and transcribed. It is prominent in metabolically active cells such as neurons and liver cells. Heterochromatin predominates in metabolically inactive cells such as small circulating lymphocytes and sperm or in cells that produce one major product such as plasma cells. The smallest units of chromatin structure are macromolecular complexes of DNA and histones called nucleosomes.


Histology_Ross

Histology_Ross. The smallest units of chromatin structure are macromolecular complexes of DNA and histones called nucleosomes. Nucleosomes are found in both euchromatin and heterochromatin and in chromosomes. These 10-nm-diameter particles represent the first level of chromatin folding and are formed by the coiling of the DNA molecule around a protein core. This step shortens the DNA molecule by approximately sevenfold relative to the unfolded DNA molecule. The core of the nucleosome consists of eight histone molecules (called an octamer). Two loops of DNA (approximately 146 nucleotide pairs) are wrapped around the core octomer. The DNA extends between each particle as a 2-nm filament that joins adjacent nucleosomes. When chromatin is extracted from the nucleus, the nucleosomal substructure of chromatin is visible in transmission electron microscopy (TEM) and is often described as “beads on a string” (Fig. 3.2a).


Histology_Ross

Histology_Ross. In the next step, a long strand of nucleosomes is coiled to produce a 30-nm chromatin fbril. Six nucleosomes form one turn in the coil of the chromatin fibril, which is approximately 40-fold shorter than unfolded DNA. Long stretches of 30-nm chromatin fibrils are further organized into loop domains (containing 15,000 to 100,000 base pairs), which are anchored into a chromosome scaffold or nuclear matrix composed of nonhistone proteins. In heterochromatin, the chromatin fibers are tightly packed and folded on each other; in euchromatin, the chromatin fibrils are more loosely arranged. In dividing cells, chromatin is condensed and organized into discrete bodies called chromosomes.


Histology_Ross

Histology_Ross. In dividing cells, chromatin is condensed and organized into discrete bodies called chromosomes. During mitotic division, chromatin fbers formed from chromatin loop domains attached to a flexible protein scaffold undergo condensation to form chromosomes [Gr., colored bodies]. Each chromosome is formed by two chromatids that are joined together at a point called the centromere (Fig. 3.2b). The double nature of the chromosome is produced in the preceding synthetic (S) phase of the cell cycle that maintains telomere length. For example, in cells that (see page 87), during which DNA is replicated in anticipation have been transformed into malignant cells, an enzyme of the next mitotic division. called telomerase is present that adds repeated nucleotide


Histology_Ross

Histology_Ross. FIGURE 3.1 • Electron micrographs of nuclei from two different cell types. The large electron micrograph shows the nucleus of a nerve cell. Two nucleoli are included in the plane of section. The nucleus of this active cell, exclusive of the nucleoli, comprises almost entirely extended chromatin or euchromatin. 10,000. Inset. The smaller nucleus belongs to a circulating lymphocyte (the entire cell is shown in the micrograph). It is a relatively inactive cell. Note the paucity of cytoplasm and cytoplasmic organelles. The chromatin in the nucleus is largely condensed (heterochromatin). The lighter areas represent euchromatin. 13,000.


Histology_Ross

Histology_Ross. The area located at each end of the chromosome is called sequences to the telomere ends. Recently, expression of this the telomere. Telomeres shorten with each cell division. Re-enzyme has been shown to extend the lifespan of cells. cent studies indicate that telomere length is an important in-With the exception of the mature gametes, the egg and dicator of the lifespan of the cell. To survive indefinitely sperm, human cells contain 46 chromosomes organized as 23 (become “immortalized”), cells must activate a mechanism homologous pairs (each chromosome in the pair has the chromatin fiber with loops of chromatin fibril


Histology_Ross

Histology_Ross. FIGURE 3.2 • Packaging of chromatin into the chromosomal structure. a. Sequential steps in the packaging of nuclear chromatin are shown in this diagram, beginning with the DNA double helix and ending with the highly condensed form found in chromosomes. b. Structure of human metaphase chromosome 2 as visible in atomic force microscopic image. 20,000. (Courtesy of Dr. Tatsuo Ushiki.) same shape and size). Twenty-two pairs have identical chromosomes (i.e., each chromosome of the pair contains the same portion of the genome) and are called autosomes. The twenty-third pair of chromosomes are the sex chromosomes, designated X and Y. Females contain two X chromosomes; males contain one X and one Y chromosome. The chromosomal number, 46, is found in most of the somatic cells of the body and is called the diploid (2n) number. To simplify the description of chromosomal number and DNA changes during mitosis and meiosis, we use the lower case letter (n) for chromosome number and lower case letter


Histology_Ross

Histology_Ross. the diploid (2n) number. To simplify the description of chromosomal number and DNA changes during mitosis and meiosis, we use the lower case letter (n) for chromosome number and lower case letter (d) for DNA content. Diploid chromosomes have the (2d) amount of DNA immediately after cell division but have twice that amount—that is, the (4d) amount of DNA—after the S phase (see page 89).


Histology_Ross

Histology_Ross. As a result of meiosis, eggs and sperm have only 23 chromosomes, the haploid (1n) number, as well as the haploid (1d) amount of DNA. The somatic chromosome number (2n) and the diploid (2d) amount of DNA are reestablished at fertilization by the fusion of the sperm nucleus with the egg nucleus. In a karyotype, chromosome pairs are sorted according to their size, shape, and emitted fluorescent color.


Histology_Ross

Histology_Ross. A preparation of chromosomes derived from mechanically ruptured, dividing cells that are then fixed, plated on a microscope slide, and stained is called a metaphase spread. In the past, chromosomes were routinely stained with Giemsa stain; however, with the recent development of in situ hybridization techniques, the fluorescent in situ hybridization (FISH) procedure is now more often used to visualize a chromosomal spread. These spreads are observed with fluorescence microscopes, and computer-controlled cameras are then used to capture images of the chromosome pairs. Image-processing software is used to sort the chromosome pairs according to their morphology to form a karyotype (see Fig. F3.1.1a). A variety of molecular probes that are now commercially available are used in cytogenetic testing to diagnose disorders caused by chromosomal abnormalities such as nondisjunctions, transpositions (see Fig. F3.1.1a), deletions (see Fig. F3.1.1b), and duplications of specifc gene sites.


Histology_Ross

Histology_Ross. testing to diagnose disorders caused by chromosomal abnormalities such as nondisjunctions, transpositions (see Fig. F3.1.1a), deletions (see Fig. F3.1.1b), and duplications of specifc gene sites. Karyotypes are also used for prenatal determination of sex in fetuses and for prenatal screening for certain genetic diseases (see Fig. 1.7).


Histology_Ross

Histology_Ross. The Barr body can be used to identify the sex of a fetus. Some chromosomes are repressed in the interphase nucleus and exist only in the tightly packed heterochromatic form. One X chromosome of the female is an example of such a chromosome. This fact can be used to identify the sex of a fetus. This chromosome was discovered in 1949 by Barr and Bartram in nerve cells of female cats, where it appears as a well-stained round body, now called the Barr body, adjacent to the nucleolus.


Histology_Ross

Histology_Ross. Although the Barr body was originally found in sectioned tissue, it was subsequently shown that any relatively large number of cells prepared as a smear (e.g., scrapings of the oral mucous membrane from the inside of the cheeks or neutrophils from a blood smear) can be used to search for the Barr body. In cells of the oral mucous membrane, the Barr body is located adjacent to the nuclear envelope. In neutrophils, the Barr body forms a drumstick-shaped appendage on one of the nuclear lobes (Fig. 3.3). In both sections and smears, many cells must be examined to find those whose orientation is suitable for the display of the Barr body. FIGURE 3.3 • Photomicrograph of a neutrophil from a female patient’s blood smear. The second X chromosome of the female patient is repressed in the interphase nucleus and can be demonstrated in the neutrophil as a drumstick-appearing appendage (arrow) on a nuclear lobe. 250.


Histology_Ross

Histology_Ross. The nucleolus is the site of ribosomal RNA (rRNA) synthesis and initial ribosomal assembly. The nucleolus is a nonmembranous region of the nucleus that surrounds transcriptionally active rRNA genes. It is the primary site of ribosomal production and assembly. The nucleolus varies in size but is particularly well developed in cells active in protein synthesis. Some cells contain more than one nucleolus (Fig. 3.4). The nucleolus has three morphologically distinct regions:  Fibrillar centers contain DNA loops of five different chromosomes (13, 14, 15, 21, and 22) that contain rRNA genes, RNA polymerase I, and transcription factors.  Fibrillar material (pars fbrosa) contain ribosomal genes that are actively undergoing transcription and large amounts of rRNA.  Granular material (pars granulosa) represents the site of initial ribosomal assembly and contains densely packed preribosomal particles.


Histology_Ross

Histology_Ross.  Granular material (pars granulosa) represents the site of initial ribosomal assembly and contains densely packed preribosomal particles. The network formed by the granular and the fibrillar materials is called the nucleolonema. rRNA is present in both granular and fibrillar material and is organized, respectively, as both granules and extremely fine filaments packed tightly together. Genes for the ribosomal subunits are localized in FIGURE 3.4 • Electron micrograph of the nucleolus. This nucleolus from a nerve cell shows fibrillar centers (FC) surrounded by the fibrillar (F) and granular (G) materials. Such a network of both materials is referred to as the nucleolonema. The rRNA, DNA-containing genes for the rRNA, and specific proteins are localized in the interstices of the nucleolonema. 15,000.


Histology_Ross

Histology_Ross. the interstices of this network and are transcribed by RNA polymerase I. After further processing and modification of rRNA by small nucleolar RNAs (snoRNAs), the subunits of rRNA are assembled using ribosomal proteins imported from the cytoplasm. The partially assembled ribosomal subunits (preribosomes) are exported from the nucleus via nuclear pores for full assembly into mature ribosomes in the cytoplasm. The nucleolus is involved in regulation of the cell cycle.


Histology_Ross

Histology_Ross. The nucleolus is involved in regulation of the cell cycle. Nucleostemin is a newly identified protein that has been found within the nucleolus. Nucleostemin is a p53 binding protein that regulates the cell cycle and influences cell differentiation (page 88). As cellular differentiation progresses, the level of this protein decreases. The presence of nucleostemin in malignant cells suggests that it could play a role in their uncontrolled proliferation (Folder 3.2). In addition, DNA, RNA, and retroviruses and their viral proteins interact with the nucleolus and cause redistribution of fbrillar and granular materials during the course of viral infection. These viruses can use components of the nucleolus as part of their own replication process. Evidence suggests that viruses may target the nucleolus and its components to favor viral transcription and translation and perhaps alter the cell cycle to promote viral replication.


Histology_Ross

Histology_Ross. The nucleolus stains intensely with hematoxylin and basic dyes and metachromatically with thionine dyes. That the basophilia and metachromasia of the nucleolus are related to the phosphate groups of the nucleolar RNA is confirmed by predigestion of specimens with ribonuclease (RNAse), which abolishes the staining. As mentioned above, DNA is present in the nucleolus; however, its concentration is below the detection capability of the Feulgen reaction. Thus, when examined in the light microscope, nucleoli


Histology_Ross

Histology_Ross. Cytogenetic testing is an important component in the di-agnosis and evaluation of genetic disorders and refers to the analysis of chromosomes. Chromosome abnormalities occur in approximately 0.5% of all live births and are de-tected in about 50% of the first trimester miscarriages (spontaneous abortions) and about 95% of various tumor cells. Chromosome analysis can be performed on periph-eral blood, bone marrow, tissues (such as skin or chorionic villi obtained from biopsies), and cells obtained from amni-otic fluid during amniocentesis. Studies of chromosomes begin with the extraction of whole chromosomes from the nuclei of dividing cells. These chromosomes are then placed on glass slides, hybridized with special fluorescence probes (FISH tech-nique), and examined under a microscope. A single fluo-rescent DNA probe produces a bright microscopic signal when the probe is hybridized to a specific part of a partic-ular chromosome. To obtain an image of all of the chromo-somes, a mixture


Histology_Ross

Histology_Ross. fluo-rescent DNA probe produces a bright microscopic signal when the probe is hybridized to a specific part of a partic-ular chromosome. To obtain an image of all of the chromo-somes, a mixture of different probes is used to produce dif-ferent colors in each chromosome. Karyotypes labeled by this method allow cytogeneticists to perform a comprehen-sive analysis of changes in the number of chromosomes and chromosomal abnormalities such as additions or dele-tions. The paired chromosomes are numbered in the kary-otype, and the male sex is indicated by the presence of chromosomes X and Y (see Fig. F3.1.1a). The white box inset in Figure F3.1.1a shows the XX chromosome pair as it appears in the female. Sometimes, part of a chromosome will break off and at-tach to another chromosome. When this happens, it is re-ferred to as a translocation. Note that the red box inset in Figure F3.1.1a shows a translocation between chromo-some 8 and 14 (t8;14). It is clearly visible on this color image that


Histology_Ross

Histology_Ross. it is re-ferred to as a translocation. Note that the red box inset in Figure F3.1.1a shows a translocation between chromo-some 8 and 14 (t8;14). It is clearly visible on this color image that a part of the original chromosome 8 (aqua blue region) is now attached to chromosome 14, and a small portion of chromosome 14 (red region) is now part of


Histology_Ross

Histology_Ross. FOLDER 3.1 Clinical Correlation: Cytogenetic Testing FIGURE F3.1.1 • Karyotypes obtained with the FISH technique. a. Karyotype of a normal male. The white box inset shows the XX chromosome pair of a normal female. The red box inset reveals an abnormality in chromosomes 14 and 8. (Courtesy of the Applied Imaging International Ltd., Newcastle upon Tyne, UK.) b. A metaphase spread from a patient with Prader-Willi/Angelman syndrome. The yellow box inset shows the enlarged pair of chromosome 15. (Courtesy of Dr. Robert B. Jenkins.) 1 6 7 8 8 9 10 11 12 13 19 20 21 22 X XX Y 14 14 15 16 17 18 2 3 4 5 15 baba appear Feulgen-negative with Feulgen-positive nucleolus-associated chromatin that often rims the nucleolus. The nuclear envelope, formed by two membranes with a perinuclear cisternal space between them, separates the nucleoplasm from the cytoplasm.


Histology_Ross

Histology_Ross. The nuclear envelope provides a selectively permeable membranous barrier between the nuclear compartment and the cytoplasm, and it encloses the chromatin. The nuclear envelope is assembled from two (outer and inner) nuclear membranes with a perinuclear cisternal space between them. The peri nuclear clear cisternal space is continuous with the cisternal space of the rER (Fig. 3.5). The two membranes of the envelope are perforated at intervals by nuclear pores that mediate the active transport of proteins, ribonucleoproteins, and RNAs between the nucleus and cytoplasm. The membranes of the nuclear envelope differ in structure and functions:  The outer nuclear membrane closely resembles the membrane of the endoplasmic reticulum and in fact is continuous with rER membrane (see Fig 3.5). Polyribosomes are often attached to ribosomal docking proteins present on the cytoplasmic side of the outer nuclear membrane.


Histology_Ross

Histology_Ross.  The inner nuclear membrane is supported by a rigid network of intermediate protein filaments attached to its inner surface called the nuclear (fbrous) lamina. In addition, the inner nuclear membrane contains specific lamin receptors and several lamina-associated proteins that bind to chromosomes and secure the attachment of the nuclear lamina. The nuclear lamina is formed by intermediate filaments and lies adjacent to the inner nuclear membrane. The nuclear lamina, a thin, electron-dense intermediate filament networklike layer, resides underneath the nuclear FOLDER 3.2 Clinical Correlation: Regulation of Cell Cycle and Cancer Treatment


Histology_Ross

Histology_Ross. Understanding the details of cell-cycle regulation has had an impact on cancer research and has contributed to the development of new treatments. For instance, inactiva-tion of tumor-suppressor genes has been shown to play a role in the growth and division of cancer cells. The pro-teins encoded by these genes are used by the cell throughout several DNA-damage checkpoints. For in-stance, mutations in the breast cancer susceptibility gene 1 (BRCA-1) and breast cancer susceptibility gene 2 (BRCA-2) are associated with an increased risk for bilateral breast cancer. Both protein products of these tumor-suppressor genes–namely, BRCA-1 and BRCA-2 proteins–are directly involved in multiple cellular pro-cesses in response to DNA damage, including checkpoint activation, gene transcription, and repair of DNA double-strand breaks. Together with RAD-51 protein, which is involved in the homologous recombination and repair of DNA, they maintain stability of the human genome. The defective BRCA


Histology_Ross

Histology_Ross. repair of DNA double-strand breaks. Together with RAD-51 protein, which is involved in the homologous recombination and repair of DNA, they maintain stability of the human genome. The defective BRCA proteins are unable to interact with RAD-51. By screening patients for mutations in these genes, much earlier detection of cancer can be accomplished. It is also now known why in some individuals p53 mu-tations make their tumors resistant to radiotherapy. DNA damage caused by therapeutic radiation procedures is de-tected by DNA-damage checkpoints, which cause cancer cells to be arrested in the cell cycle. However, these cells will not die because of the absence of functional p53, which triggers apoptosis.


Histology_Ross

The Cell Nucleus NUCLEAR COMPONENTS

Histology_Ross. The Cell Nucleus NUCLEAR COMPONENTS


Histology_Ross

Histology_Ross. FIGURE 3.5 • Structure of the nuclear envelope and its relationship to the rER. a. The nuclear wall consists of a double membrane envelope that surrounds the nucleus. The outer membrane is continuous with the membranes of the rER; thus, the perinuclear space communicates with the rER lumen. The inner membrane is adjacent to nuclear intermediate filaments that form the nuclear lamina. b. This electron micrograph, prepared by the quick-freeze deep-etch technique, shows the nucleus, the large spherical object, surrounded by the nuclear envelope. Note that the outer membrane possesses ribosomes and is continuous with the rER. 12,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.) membrane. It addition to its supporting or “nucleoskeletal” function, nuclear lamina is essential in many nuclear activities such as DNA replication, transcription, and gene regulation. If the membranous component of the nuclear envelope is disrupted by exposure to detergent, the


Histology_Ross

Histology_Ross. is essential in many nuclear activities such as DNA replication, transcription, and gene regulation. If the membranous component of the nuclear envelope is disrupted by exposure to detergent, the nuclear lamina remains, and the nucleus retains its shape.


Histology_Ross

Histology_Ross. The major components of the lamina, as determined by biochemical isolation, are nuclear lamins, a specialized type of nuclear intermediate filament (see page 63), and laminassociated proteins. Nuclear lamina is essentially composed of lamin A and lamin C proteins that form intermediate filaments. These filaments are cross-linked into an orthogonal lattice (Fig. 3.6), which is attached mainly via lamin B protein to the inner nuclear membrane through its interactions with lamin receptors. The family of lamin receptors includes emerin (34 kilodaltons) that binds both lamin A and B, nurim (29 kilodaltons) that binds lamin A, and a 58-kilodalton lamin B receptor (LBR) that, as its name suggests, binds lamin B.


Histology_Ross

Histology_Ross. Unlike other cytoplasmic intermediate filaments, lamins disassemble during mitosis and reassemble when mitosis ends. The nuclear lamina appears to serve as scaffolding for chromatin, chromatin-associated proteins, nuclear pores, and the membranes of the nuclear envelope. In addition, it is involved in nuclear organization, cell-cycle regulation, differentiation, and gene expression.


Histology_Ross

Histology_Ross. Impairment in nuclear lamina architecture or function is associated with certain genetic diseases (laminopathies) and apoptosis. Mutations in lamin A/C cause tissue-specifc diseases that affect striated muscle, adipose tissue, peripheral nerve or skeletal development, and premature aging. Recently, two hereditary forms of Emery-Dreifuss muscular dystrophy (EDMD) have been associated with mutations in either lamins or lamin receptors. The X-linked recessive form of EDMD is caused by mutations of emerin, while the autosomal dominant form of EDMD is caused by mutations in lamin A/C. In general, EDMD is characterized by an early-onset contractures of major tendons, very slow progressive muscle weakness, muscle wasting in the upper and lower limbs, and cardiomyopathy (weakening of the heart muscle). The nuclear envelope has an array of openings called nuclear pores.


Histology_Ross

Histology_Ross. The nuclear envelope has an array of openings called nuclear pores. At numerous sites, the paired membranes of the nuclear envelope are punctuated by 70to 80-nm “openings” through the envelope. These nuclear pores are formed from the merging of the inner and outer membranes of the nuclear envelope. With an ordinary TEM, a diaphragmlike structure appears to cross the pore opening (Fig. 3.7). Often a small dense body is observed in the center of the opening (Fig. 3.8). Since such profiles are thought to represent either ribosomes or other protein complexes (transporters) captured during their passage through the pore at the time of fixation, the term central plug/transporter is commonly used to describe this feature. With special techniques—such as negative staining and high-voltage transmission electron microscopy, or recently,


Histology_Ross

Histology_Ross. FIGURE 3.6 • Structure of the nuclear lamina. a. This schematic drawing shows the structure of the nuclear lamina adjacent to the inner nuclear membrane. The cut window in the nuclear lamina shows the DNA within the nucleus. Note that the nuclear envelope is pierced by nuclear pore complexes, which allow for selective bidirectional transport of molecules between nucleus and cytoplasm. b. Electron micrograph of a portion of the nuclear lamina from a Xenopus oocyte. It is formed by intermediate filaments (lamins) that are arranged in a square lattice. 43,000. (Adapted from Aebi U, Cohn J, Buhle L, Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature 1986;323:560–564.) nuclear lamina DNAnucleus reticulum perinuclear space nuclear pore complex a bb cryoelectron tomography—the nuclear pore exhibits additional structural detail (see Fig. 3.8). Eight multidomain protein subunits arranged in an octagonal central framework at the periphery of each pore form a


Histology_Ross

Histology_Ross. tomography—the nuclear pore exhibits additional structural detail (see Fig. 3.8). Eight multidomain protein subunits arranged in an octagonal central framework at the periphery of each pore form a cylinderlike structure known as the nuclear pore complex (NPC). The NPC, which has an estimated total mass of 125 106 daltons, is composed of about 50 different nuclear pore complex proteins collectively referred to as nucleoporins (Nup proteins). This central framework is inserted between the cytoplasmic ring and the nuclear ring (Fig. 3.9). From the cytoplasmic ring, eight short protein fbrils protrude into the cytoplasm and point toward the center of the structure. The nucleoplasmic ring complex anchors a nuclear basket (or nuclear “cage” that resembles a fish trap) assembled from eight thin 50-nm-long filaments joined distally by an adjustable terminal ring 30 to 50 nm in diameter (see Fig. 3.9). The cylinder-shaped central framework encircles the central pore of the NPC, which acts as


Histology_Ross

Histology_Ross. 50-nm-long filaments joined distally by an adjustable terminal ring 30 to 50 nm in diameter (see Fig. 3.9). The cylinder-shaped central framework encircles the central pore of the NPC, which acts as a close-fitting diaphragm or gated channel. In addition, each NPC contains one or more water-filled channels for transport of small molecules.


Histology_Ross

Histology_Ross. The NPC mediates bidirectional nucleocytoplasmic transport.


Histology_Ross

Histology_Ross. Various experiments have shown that the NPC regulates the passage of proteins between the nucleus and the cytoplasm. The significance of the NPC can be readily appreciated, as the nucleus does not carry out protein synthesis. Ribosomal proteins are partially assembled into ribosomal subunits in the nucleolus and are transported through nuclear pores to the cytoplasm. Conversely, nuclear proteins, such as histones and lamins, are produced in the cytoplasm and are transported through nuclear pores into the nucleus. Transport through the NPC largely depends on the size of the molecules:  Large molecules (such as large proteins and macromolecular complexes) depend on the presence of an attached signal sequence called the nuclear localization signal (NLS) for passage through the pores. Labeled NLS proteins destined for the nucleus then bind to a soluble cytosolic receptor called a nuclear import receptor (importin) that directs them from the cytoplasm to an appropriate NPC.


Histology_Ross

Histology_Ross. FIGURE 3.7 • Electron micrograph of the nuclear envelope. Note the visible nuclear pore complexes (arrows) and the two membranes that constitute the nuclear envelope. At the periphery of each pore, the outer and inner membranes of the nuclear envelope appear continuous. 30,000. They are then actively transported through the pore by a GTP energy-dependent mechanism. An export of proteins and RNA from the nucleus is similar to the import mechanism into the nucleus. Proteins that possess the nuclear export sequence (NES) bind in the nucleus to exportin (a protein that moves molecules from nucleus into cytoplasm) and to a GTP molecule. Protein–exportin–GTP complexes pass through NPC into the cytoplasm where GTP is hydrolyzed and the NES protein is released. The NPC transports proteins, all forms of RNA, as well as ribosomal subunits in their fully folded configurations.


Histology_Ross

Histology_Ross.  Ions and smaller water-soluble molecules (less than 9 daltons) may cross the water-flled channels of the NPC by simple diffusion. This process is nonspecific and does not require nuclear signal proteins. The effective size of the pore is about 9 nm for substances that cross by diffusion rather than the 70to 80-nm measurement of the pore boundary. However, even the smaller nuclear proteins that are capable of diffusion are selectively transported, presumably because the rate is faster than simple diffusion. During cell division, the nuclear envelope is disassembled to allow chromosome separation and is later reassembled as the daughter cells form.


Histology_Ross

Histology_Ross. During cell division, the nuclear envelope is disassembled to allow chromosome separation and is later reassembled as the daughter cells form. In late prophase of cell division, enzymes (kinases) are activated that cause phosphorylation of the nuclear lamins and other lamina-associated proteins of the nuclear envelope. After phosphorylation, the proteins become soluble, and the nuclear envelope disassembles. The lipid component of the nuclear membranes then disassociates from the proteins and is retained in small cytoplasmic vesicles. The replicated chromosomes then attach to the microtubules of the mitotic spindle and undergo active movement.


Histology_Ross

Histology_Ross. Reassembly of the nuclear envelope begins in late anaphase, when phosphatases are activated to remove the phosphate residues from the nuclear lamins. During telophase, the nuclear lamins begin to repolymerize and form the nuclear lamina material around each set of daughter chromosomes. At the same time, vesicles containing the lipid components of the nuclear membranes and structural membrane protein components fuse, and an envelope is formed on the surface of the already-reassembled nuclear lamina. By the end of telophase, formation of a nuclear envelope in each daughter cell is complete. Nucleoplasm is the material enclosed by the nuclear envelope exclusive of the chromatin and the nucleolus.


Histology_Ross

Histology_Ross. Nucleoplasm is the material enclosed by the nuclear envelope exclusive of the chromatin and the nucleolus. Although crystalline, viral, and other inclusions are sometimes found in the nucleoplasm, until recently, morphologic techniques showed it to be amorphous. It must be assumed, however, that many proteins and other metabolites reside in or pass through the nucleus in relation to the synthetic and metabolic activity of the chromatin and nucleolus. New structures have recently been identified within the nucleoplasm, including intranuclear lamin-based arrays, the protein filaments emanating inward from the nuclear pore complexes, and the active gene-tethered RNA transcription and processing machinery itself. Somatic cells in the adult organism may be classified according to their mitotic activity.


Histology_Ross

Histology_Ross. Somatic cells in the adult organism may be classified according to their mitotic activity. The level of mitotic activity in a cell can be assessed by the number of mitotic metaphases visible in a single high-magnification light microscopic field or by autoradiographic studies of the incorporation of tritiated thymidine into the newly synthesized DNA before mitosis. Using these methods, cell populations may be classified as static, stable, or renewing. FIGURE 3.8 • Cryoelectron tomography of the nuclear pore complex. These surface renderings of electron tomograms obtained from the frozen-hydrated Dictyostelium nuclei show detailed structure of the nuclear pore complex (NPC). 320,000.


Histology_Ross

Histology_Ross. a. Cytoplasmic face of the NPC shows eight protein fibrils arranged around the central channel. They protrude from the cytoplasmic ring subunits and point toward the center of the structure. Note a presence of the central plug or transporter within the central pore, which represents either ribosomes or other protein transporters captured during their passage through the NPC. b. Nuclear face of the NPC shows the nucleoplasmic ring subunits connected by nuclear filaments with the basket indicated in brown color. (Adapted from Beck M, Fster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, Baumeister W, Medalia, O. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 2004;306:1387–1390.)


Histology_Ross

Histology_Ross. FIGURE 3.9 • Sagittal section of the nuclear pore complex. Cryoelectron tomographic view of a sagittal section of the nuclear pore complex shown in Figure 3.8 is compared with a schematic drawing of the complex. Note that the central plug/transporter has been removed from the central pore. 320,000. Each pore contains eight protein subunits arranged in an octagonal central framework at the periphery of the pore. These subunits form a nuclear pore complex that is inserted between two cytoplasmic and nucleoplasmic rings. Eight short protein fibrils protrude from the cytoplasmic rings into the cytoplasm. The nuclear ring anchors a basket assembled from eight thin filaments joined distally into terminal ring which diameter can be adjusted to meet nuclear pore transport requirements. The cylindrical central framework encircles the central pore, which acts as a close-fitting diaphragm. (Adapted from Beck M, Frster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, Baumeister W, Medalia, O.


Histology_Ross

Histology_Ross. cylindrical central framework encircles the central pore, which acts as a close-fitting diaphragm. (Adapted from Beck M, Frster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, Baumeister W, Medalia, O. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 2004;306:1387–1390.)  Static cell populations consist of cells that no longer divide (postmitotic cells), such as cells of the central nervous system and skeletal or cardiac muscle cells. Under certain circumstances some of these cells (i.e., cardiac myocytes) may enter mitotic division.


Histology_Ross

Histology_Ross.  Stable cell populations consist of cells that divide episodically and slowly to maintain normal tissue or organ structure. These cells may be stimulated by injury to become more mitotically active. Periosteal and perichondrial cells, smooth muscle cells, endothelial cells of blood vessels, and fibroblasts of the connective tissue may be included in this category.  Renewing cell populations may be slowly or rapidly renewing but display regular mitotic activity. Division of such cells usually results in two daughter cells that differentiate both morphologically and functionally or two cells that remain as stem cells. Daughter cells may divide one or more times before their mature state is reached. The differentiated cell may ultimately be lost from the body.


Histology_Ross

Histology_Ross.  Slowly renewing populations include smooth muscle cells of most hollow organs, fibroblasts of the uterine wall, and epithelial cells of the lens of the eye. Slowly renewing populations may actually slowly increase in size during life, as do the smooth muscle cells of the gastrointestinal tract and the epithelial cells of the lens.  Rapidly renewing populations include blood cells, epithelial cells and dermal fibroblasts of the skin, and the epithelial cells and subepithelial fibroblasts of the mucosal lining of the alimentary tract. Phases and Checkpoints Within the Cell Cycle The cell cycle represents a self-regulated sequence of events that controls cell growth and cell division.


Histology_Ross

Histology_Ross. For renewing cell populations and growing cell populations, including embryonic cells, and cells in tissue culture, the goal of the cell cycle is to produce two daughter cells, each containing chromosomes identical to those of the parental cell. The cell cycle incorporates two principal phases: the interphase, representing continuous growth of the cell, and the M phase (mitosis), characterized by the partition of the genome. Three other phases, G1 (gap1) phase, S (synthesis) phase, and G2 (gap2) phase, further subdivide interphase (Fig. 3.10). Rapidly renewing populations of human cells progress through the full cell cycle in about 24 hours. Throughout the cycle, several internal quality-control mechanisms or checkpoints represented by biochemical pathways control transition between cell-cycle stages. The cell cycle stops at several checkpoints and can only proceed if certain conditions are met—for example, if the cell has reached a certain size. Checkpoints monitor and modulate the


Histology_Ross

Histology_Ross. stages. The cell cycle stops at several checkpoints and can only proceed if certain conditions are met—for example, if the cell has reached a certain size. Checkpoints monitor and modulate the progression of cells through the cell cycle in response to intracellular or environmental signals.


Histology_Ross

Histology_Ross. The G1 phase is usually the longest and the most variable phase of the cell cycle, and it begins at the end of M phase. During the G1 phase, the cell gathers nutrients and synthesizes RNA and proteins necessary for DNA synthesis and 912 h 7.510 h 3.54.5 h 1 h G1 GO GTD S G2 M G1 DNA-damage checkpoint G2 DNA-damage checkpoint S DNA-damage checkpoint unreplicated DNA checkpoint checkpoint checkpoint restriction checkpoint


Histology_Ross

Histology_Ross. FIGURE 3.10 • Cell cycle and checkpoints. This diagram illustrates the cell cycle of rapidly dividing cells in relation to DNA synthesis. After mitosis, the cell is in interphase. G1 represents the period during which a gap occurs in DNA synthesis. S represents the period during which DNA synthesis occurs. G2 represents a second gap in DNA synthesis. GO represents the path of a cell that has stopped dividing; however, such a cell may reenter the cell cycle after an appropriate stimulus. The cell residing in GO may undergo terminal differentiation, GTD, and produce a population of permanent nondividing cells (e.g., mature fat cells). The average timing of each phase of the cell cycle is indicated on the diagram. Each phase contains several checkpoints that ensure that the system only proceeds to the next stage when the previous stage has been completed and no damage to the DNA is detected.


Histology_Ross

Histology_Ross. chromosome replication. The cell’s progress through this phase is monitored by two checkpoints: (1) the restriction point, which is sensitive to the size of the cell, the state of the cell’s physiologic processes, and its interactions with extracellular matrix; and (2) the G1 DNA-damage checkpoint, which monitors the integrity of newly replicated DNA. For instance, if the DNA has irreparable damage, then the G1 DNA-damage checkpoint detects the high levels of tumor-suppressing protein p53 and it does not allow the cell to enter the S phase. The cell will then most likely undergo programmed cell death (apoptosis).


Histology_Ross

Histology_Ross. The restriction point (or “point of no return”) is the most important checkpoint in the cell cycle. At this checkpoint, the cell self-evaluates its own replicative potential before deciding to either enter the S phase and the next round of cell division or to retire and leave the cell cycle. A cell that leaves the cycle in the G1 phase usually begins terminal differentiation by entering the GO phase (“O” stands for “outside” the cycle). Thus, the G1 phase may last for only a few hours (average 9 to 12 hours) in a rapidly dividing cell, or it may last a lifetime in a nondividing cell. This checkpoint is mediated by interactions between the retinoblastoma susceptibility protein (pRb) and a family of essential transcription factors (E2F) with target promoters. In normal cells, proper interaction between pRb and E2F turns off many genes and blocks cell-cycle progression. In the S phase, DNA is replicated.


Histology_Ross

Histology_Ross. In the S phase, DNA is replicated. Initiation of DNA synthesis marks the beginning of the S phase, which is about 7.5 to 10 hours in duration. The DNA of the cell is doubled during the S phase, and new chromatids are formed that will become obvious at prophase or metaphase of the mitotic division. Chromosome replication is initiated at many different sites called replicons along the chromosomal DNA. Each replicon has a specifically assigned time frame for replication during S phase. Presence of the S DNA-damage checkpoint in this phase monitors quality of replicating DNA. In the G2 phase, the cell prepares for cell division.


Histology_Ross

Histology_Ross. In the G2 phase, the cell prepares for cell division. During this phase, the cell examines its replicated DNA in preparation for cell division. This is a period of cell growth and reorganization of cytoplasmic organelles before entering the mitotic cycle. The G2 phase may be as short as 1 hour in rapidly dividing cells or of nearly indefinite duration in some polyploid cells and in cells such as the primary oocyte that are arrested in G2 for extended periods. Two checkpoints monitor DNA quality: the G2 DNA-damage checkpoint and the unreplicated-DNA checkpoint. The latter checkpoint prevents the progression of the cell into the M phase before DNA synthesis is complete. Mitosis occurs in the M phase.


Histology_Ross

Histology_Ross. Mitosis occurs in the M phase. Mitosis nearly always includes both karyokinesis (division of the nucleus) and cytokinesis (division of the cell) and lasts about 1 hour. Mitosis takes place in several stages described in more detail below. Separation of two identical daughter cells concludes the M phase. The M phase possesses two checkpoints: the spindle-assembly checkpoint, which prevents premature entry into anaphase, and the chromosome-segregation checkpoint, which prevents the process of cytokinesis until all of the chromosomes have been correctly separated. The mitotic catastrophe caused by malfunction of cell-cycle checkpoints may lead to cell death and tumor cell development.


Histology_Ross

Histology_Ross. The mitotic catastrophe caused by malfunction of cell-cycle checkpoints may lead to cell death and tumor cell development. Malfunction of any of the three DNA-damage checkpoints at the G1, S, and G2 phases of the cell cycle and the spindle-assembly checkpoint at M phase may lead to a mitotic catastrophe. Mitotic catastrophe is defined as the failure to arrest the cell cycle before or at mitosis, resulting in aberrant chromosome segregation. Under normal conditions, death in these cells will occur by activation of the apoptotic cycle. Cells that fail to execute the apoptotic cycle in response to DNA or mitotic spindle damage are likely to divide asymmetrically in the next round of cell division. This leads to the generation of aneuploid cells (cells containing abnormal chromosome numbers). Thus, a mitotic catastrophe may be regarded as one of the mechanisms contributing to oncogenesis (tumor cell development).


Histology_Ross

Histology_Ross. Malfunction of the restriction checkpoint at the G1 phase may also result in malignant transformation of cells. Malignant cells lose contact inhibition, a normal process in which cells inhibit their division when they contact other cells. Malignant cells in culture continue to divide and may grow on top of one another rather then discontinuing growth when the plate is fully covered in a monolayer of cells. The malfunction of the restriction checkpoint may be facilitated by the viral proteins of several cancer-causing viruses, such as the T-antigen of simian virus (SV40) that binds to pRb. This binding alters the configuration of the pRb–T-antigen complex and renders the restriction checkpoint inoperable, thus facilitating the cell’s progression from the G1 to S phase of the cell cycle. This mechanism of carcinogenesis occurs in mesothelioma (cancer of the lining epithelium of the pleural cavities in the thorax), osteosarcoma (a type of bone cancer), and ependymoma (a type of childhood


Histology_Ross

Histology_Ross. mechanism of carcinogenesis occurs in mesothelioma (cancer of the lining epithelium of the pleural cavities in the thorax), osteosarcoma (a type of bone cancer), and ependymoma (a type of childhood brain tumor).


Histology_Ross

Histology_Ross. The reserve stem cell population may become activated and reenter the cell cycle. Cells identified as reserve stem cells may be thought of as GO cells that may be induced to reenter the cell cycle in response to injury of cells within the tissues of the body. Activation of these cells may occur in normal wound healing and in repopulation of the seminiferous epithelium after intense acute exposure of the testis to X-irradiation or during regeneration of an organ, such as the liver, after removal of a major portion. If damage is too severe, even the reserve stem cells die, and there is no potential for regeneration. Regulation of the Cell Cycle Passage through the cell cycle is driven by proteins that are cyclically synthesized and degraded during each cycle. A number of cytoplasmic protein complexes regulate and control the cell cycle. Some of these proteins function as


Histology_Ross

Histology_Ross. TABLE Functional Summary of Cyclin–Cdk Complexes Used in Regulating the Human Cell Cycle3.1 Associated Cyclin-Cyclin Type Dependent Protein Kinase Targeted Phase of Cell Cycle Targeted Effector Proteins Cyclin D Cdk4/6 G1 phase progression Tumor-suppressing protein p53, retinoblastoma susceptibility protein (pRb) Cyclin E Cdk2 S phase entry ATMA or ATRB protein kinases, tumor-suppressing protein p53 Cyclin A Cdk2 S phase progression Replication protein A, DNA polymerase, minichromosome maintenance (Mcm) protein Cyclin A Cdk1 S phase through G2 phase Cdc25 phosphatase, cyclin B and M phase entry Cyclin E Cdk1 M phase progression Chromatin-associated proteins, histone H1, nuclear lamins, myosin regulatory proteins, centrosomal proteins, transcription factors c-fos/jun, c-myb, oct-1, SWI5; p60src protein kinases, casein kinase II, c-mos protein kinases A Ataxia–telangiectasia mutated protein kinase B ATM—and Rad3—related kinase biochemical oscillators, whose synthesis and degradation are


Histology_Ross

Histology_Ross. protein kinases, casein kinase II, c-mos protein kinases A Ataxia–telangiectasia mutated protein kinase B ATM—and Rad3—related kinase biochemical oscillators, whose synthesis and degradation are coordinated with specific phases of the cycle. Cellular and molecular events induced during the increase and decrease of different protein levels are the basis of the cell-cycle “engine.” Other proteins actively monitor the quality of the molecular processes at the different checkpoints distributed throughout the cycle (described above). The protein complexes at the checkpoints may drive the cell into and out of the cell cycle, stimulating growth and division when conditions are favorable and, conversely, stopping or reducing the rate of cell division when conditions are not favorable.


Histology_Ross

Histology_Ross. A two-protein complex consisting of cyclin and a cyclindependent kinase (Cdk) helps power the cells through the checkpoints of cell-cycle division. The first milestone in understanding the regulation of the cell cycle was the discovery in the early 1970s of a protein called maturation promoting factor (MPF). MPF appeared to control the initiation of mitosis. When injected into the nuclei of immature frog oocytes, which are normally arrested in G2, the cells immediately proceeded through mitosis. MPF was eventually found to consist of two proteins:  Cdc2 (also known as Cdk-1), a 32-kilodalton member of the Cdk family of proteins; and  Cyclin B, a 45-kilodalton member of the cyclin family, which are key regulators of the cell cycle. Cyclins are synthe sized as constitutive proteins; however, their levels during the cell cycle are controlled by ubiquitin-mediated degradation.


Histology_Ross

Histology_Ross. It is now known that the cyclin–Cdk complex acts at different phases of the cell cycle and targets different proteins to control cell-cycle–dependent functions. Table 3.1 shows the combination of the different types of cyclins with different types of Cdks and how interactions between these two proteins affect cells progressing through the cell cycle. Passage through the cell cycle requires an increase in cyclin–Cdk activity in some phases followed by the decline of that activity in other phases (Fig. 3.11). The increased activity of FIGURE 3.11 • Regulation of the cell cycle by cyclin–Cdk complexes. This diagram shows the changing pattern of cyclin–Cdk activities during different phases of the cell cycle. cyclin–Cdk is achieved by the stimulatory action of cyclins and is counterbalanced by the inhibitory action of proteins such as Inks (inhibitors of kinase), Cips (Cdk inhibitory proteins), and Kips (kinase inhibitory proteins).


Histology_Ross

Histology_Ross. Cell division is a crucial process that increases the number of cells, permits renewal of cell populations, and allows wound repair. Mitosis is a process of chromosome segregation and nuclear division followed by cell division that produces two daughter cells with the same chromosome number and DNA content as the parent cell. The term mitosis is used to describe the equal partitioning of replicated chromosomes and their genes into two identical groups. The process of cell division includes division of both the nucleus (karyokinesis) and the cytoplasm (cytokinesis). The process of cytokinesis results in distribution of nonnuclear organelles into two daughter cells. Before entering mitosis, cells duplicate their DNA. This phase of the cell cycle is called the S or synthesis phase. At the beginning of this phase, the chromosome number is (2n), and the DNA content is also (2d); at the end, the chromosome number remains the same (2n), and the DNA content doubles to (4d).


Histology_Ross

Histology_Ross. Mitosis follows the S phase of the cell cycle and is described in four phases.


Histology_Ross

Histology_Ross. Mitosis consists of four phases (Fig. 3.12):  Prophase begins as the replicated chromosomes condense and become visible. As the chromosomes continue to condense, each of the four chromosomes derived from each homologous pair can be seen to consist of two chromatids. The sister chromatids are held together by the ring of proteins called cohesins and the centromere. In late prophase or prometaphase (sometimes identified as a separate phase of mitosis), the nuclear envelope begins to disintegrate into small transport vesicles and resembles the sER. The nucleolus, which may still be present in some cells, also completely disappears in prometaphase. In addition, a highly specialized protein complex called a kinetochore appears on each chromatid opposite to the centromere (Fig. 3.13). The protein complexes that form kinetochores in the centromere region of chromatid are attached to specific repetitive DNA sequences known as satellite DNA that are similar in each chromosome. Microtubules of


Histology_Ross

Histology_Ross. complexes that form kinetochores in the centromere region of chromatid are attached to specific repetitive DNA sequences known as satellite DNA that are similar in each chromosome. Microtubules of the developing mitotic spindle attach to the kinetochores and thus to the chromosomes.  Metaphase (Fig. 3.14) begins as the mitotic spindle, consisting of three types of microtubules, becomes organized around the microtubule-organization centers (MTOCs) located at opposite poles of the cell. The first type, the astral microtubules, are nucleated from the -tubulin rings in a starlike fashion around each MTOC (see Fig. 2.54). The second type, the polar micro-tubules, also originates from the MTOC; however, these microtubules grow away from the MTOC. The third type, the kinetochore microtubules, emanates from the MTOC to probe the cytoplasm in search of kinetochores. When a kinetochore is finally captured by a kinetochore microtubule, it is pulled toward the MTOC, where additional microtubules


Histology_Ross

Histology_Ross. from the MTOC to probe the cytoplasm in search of kinetochores. When a kinetochore is finally captured by a kinetochore microtubule, it is pulled toward the MTOC, where additional microtubules will attach. The kinetochore is capable of binding between 30 and 40 microtubules to each chromatid. In some species, kinetochore microtubules are formed by MTOCindependent mechanisms that involve kinetochores. Kinetochore microtubules and their associated motor proteins direct the movement of the chromosomes to a plane in the middle of the cell, the equatorial or metaphase plate.


Histology_Ross

Histology_Ross.  Anaphase (Fig. 3.15) begins at the initial separation of sister chromatids. This separation occurs when the cohesins that have been holding the chromatids together break down. The chromatids then begin to separate and are pulled to opposite poles of the cell by the molecular motors (dyneins) sliding along the kinetochore micro-tubules toward the MTOC.


Histology_Ross

Histology_Ross.  Telophase (Fig. 3.16) is marked by the reconstitution of a nuclear envelope around the chromosomes at each pole. The chromosomes uncoil and become indistinct except at regions that will remain condensed in the interphase nucleus. The nucleoli reappear, and the cytoplasm divides (cytokinesis) to form two daughter cells. Cytokinesis begins with the furrowing of the plasma membrane midway between the poles of the mitotic spindle. The separation at the cleavage furrow is achieved by a contractile ring consisting of a very thin array of actin filaments positioned around the perimeter of the cell. Within the ring, myosin II molecules are assembled into small filaments that interact with the actin filaments, causing the ring to contract. As the ring tightens, the cell is pinched into two daughter cells. Because the chromosomes in the daughter cells contain identical copies of the duplicated DNA, the daughter cells are genetically identical and contain the same kind and number of


Histology_Ross

Histology_Ross. two daughter cells. Because the chromosomes in the daughter cells contain identical copies of the duplicated DNA, the daughter cells are genetically identical and contain the same kind and number of chromosomes. The daughter cells are (2d) in DNA content and (2n) in chromosome number.


Histology_Ross

Histology_Ross. Meiosis involves two sequential nuclear divisions followed by cell divisions that produce gametes containing half the number of chromosomes and half the DNA found in somatic cells.


Histology_Ross

Histology_Ross. The zygote (the cell resulting from the fusion of an ovum and a sperm) and all the somatic cells derived from it are diploid (2n) in chromosome number; thus, their cells have two copies of every chromosome and every gene encoded on this chromosome. These chromosomes are called homologous chromosomes because they are similar but not identical; one set of chromosomes is of maternal origin, the other is from the male parent. The gametes, having only one member of each chromosome pair, are described as haploid (1n). During gametogenesis, reduction in chromosome number to the haploid state (23 chromosomes in humans) occurs through metaphase I continuation of continuation of meiosis as seen meiosis as seen metaphase in spermatogenesis in oogenesis (undergo apoptosis) spermatozoa *note: prophase II, anaphase II, and telophase II are not shown ovum


Histology_Ross

Histology_Ross. FIGURE 3.12 • Comparison of mitosis and meiosis in an idealized cell with two pairs of chromosomes (2n). The chromosomes of maternal and paternal origin are depicted in red and blue, respectively. The mitotic division produces daughter cells that are genetically identical to the parental cell (2n). The meiotic division, which has two components, a reductional division and an equatorial division, produces a cell that has only two chromosomes (1n). In addition, during the chromosome pairing in prophase I of meiosis, chromosome segments are exchanged, leading to further genetic diversity. It should be noted that in humans the first polar body does not divide. Division of the first polar body does occur in some species. FIGURE 3.13 • Atomic force microscopic image of the centromeric region of a human metaphase chromosome.


Histology_Ross

Histology_Ross. FIGURE 3.13 • Atomic force microscopic image of the centromeric region of a human metaphase chromosome. The facing surfaces of two sister chromatids visible on this image form the centromere, a point of junction of both chromatids. On the opposite side from the centromere, each chromatid possesses a specialized protein complex, the kinetochore, which serves as an attachment point for kinetochore microtubules of the mitotic spindle. Note that the surface of the chromosome has several protruding loop domains formed by chromatin fibrils anchored into the chromosome scaffold. 40,000. (Courtesy of Dr. Tatsuo Ushiki.) meiosis, a process that involves two successive divisions, the second of which is not preceded by an S phase. This reduction is necessary to maintain a constant number of chromosomes in a given species. Reduction in chromosome number to (1n) in the first meiotic division is followed by reduction in DNA content to the haploid (1d) amount in the second meiotic division.


Histology_Ross

Histology_Ross. FIGURE 3.15 • Mitotic spindle in anaphase. This immunofluorescent image comes from the same cell type and identical preparation as in Figure 3.13. Connections that hold the sister chromatids together break at this stage. The chromatids are then moved to opposite poles of the cell by microtubule-associated molecular motors (dyneins and kinesins) that slide along the kinetochore microtubules toward the centriole and are also pushed by the polar microtubules (visible between the separated chromosomes) away from each other, thus moving opposite poles of the mitotic spindle into the separate cells. 1,400. (Courtesy of Dr. Thomas U. Mayer.)


Histology_Ross

Histology_Ross. FIGURE 3.14 • Mitotic spindle in metaphase. Using indirect immunofluorescence techniques, the mitotic spindle in a Xenopus XL-177 cell was labeled with an antibody against -tubulin conjugated with fluorescein (green). DNA was stained blue with fluorescent DAPI stain. In metaphase, the nuclear membrane disassembles, DNA is condensed into chromosomes, and microtubules form a mitotic spindle. The action of microtubuleassociated motor proteins on the microtubules of the mitotic spindle creates the metaphase plate along which the chromosomes align in the center of the cell. 1,400. (Courtesy of Dr. Thomas U. Mayer.) During meiosis, the chromosome pair may exchange chromosome segments, thus altering the genetic composition of the chromosomes. This genetic exchange, called crossing-over, and the random assortment of each member of the chromosome pairs into haploid gametes give rise to infinite genetic diversity.


Histology_Ross

Histology_Ross. FIGURE 3.16 • Mitotic spindle in telophase. In this phase, DNA is segregated and a nuclear envelope is reconstituted around the chromosomes at each pole of the mitotic spindle. The cell divides into two during cytokinesis. In the middle of the cell, actin, septins, myosins, microtubules, and other proteins gather as the cell establishes a ring of proteins that will constrict, forming a bridge between the two sides of what was once one cell. The chromosomes uncoil and become indistinct except at regions that remain condensed in interphase. The cell types and preparation are the same as those in Figures 3.13 and 3.14. 1,400. (Courtesy of Dr. Thomas U. Mayer.) The cytoplasmic events associated with meiosis differ in the male and female.


Histology_Ross

Histology_Ross. The cytoplasmic events associated with meiosis differ in the male and female. The nuclear events of meiosis are the same in males and females, but the cytoplasmic events are markedly different. Figure 3.12 illustrates the key nuclear and cytoplasmic events of meiosis as they occur in spermatogenesis and oogenesis. The events of meiosis through metaphase I are the same in both sexes. Therefore, the figure illustrates the differences in the process as they diverge after metaphase I. In males, the two meiotic divisions of a primary spermatocyte yield four structurally identical, although genetically unique, haploid spermatids. Each spermatid has the capacity to differentiate into a spermatozoon. In contrast, in females, the two meiotic divisions of a primary oocyte yield one haploid ovum and three haploid polar bodies. The ovum receives most of the cytoplasm and becomes the functional gamete. The polar bodies receive very little cytoplasm and degenerate.


Histology_Ross

Histology_Ross. The nuclear events of meiosis are similar in males and females. Meiosis consists of two successive mitotic divisions without the additional S phase between the two divisions. During the S phase that precedes meiosis, DNA is replicated forming sister chromatids (two parallel strands of DNA) joined together by the centromere. The DNA content becomes (4d), but the chromosome number remains the same (2n). The cells then undergo a reductional division (meiosis I) and an equatorial division (meiosis II).


Histology_Ross

Histology_Ross. During meiosis I, as the name reductional division implies, the chromosome number is reduced from diploid (2n) to haploid (1n), and the amount of DNA is reduced from the (4d) to (2d). During prophase I, double-stranded chromosomes condense, and homologous chromosomes (normally one inherited from the mother and one from the father) are paired at centromeres. At this point, recombination of genetic material between the maternal and paternal chromosome pairs may occur. In metaphase I, the homologous chromosomes with their centromeres line up along the equator of the mitotic spindle and in anaphase I are separated and distributed to each daughter cell. This results in reduction of both the chromosome number (1n) and the DNA to the (2d) amount.


Histology_Ross

Histology_Ross. No DNA replication precedes meiosis II. The division during meiosis II is always equatorial because the number of chromosomes does not change. It remains at (1n), although the amount of DNA represented by the number of chromatids is reduced to (1d). During metaphase II, each chromosome aligns along the equator of the mitotic spindle, and at anaphase II sister chromatids are separated from each other. Thus, each chromosome splits into two single-stranded chromosomes that are then distributed to each haploid daughter cell. Phases in the process of meiosis are similar to the phases of mitosis. The prophase of meiosis I is an extended phase in which pairing of homologous chromosomes, synapsis (close association of homologous chromosomes), and recombination of genetic material on homologous chromosomes is observed. Prophase I is subdivided into the following five stages (see Fig. 3.12).


Histology_Ross

Histology_Ross.  Leptotene. This stage is characterized by the condensation of chromatin and by the appearance of chromosomes. Sister chromatids also condense and become connected with each other by meiosisspecifc cohesion complexes (Rec8p). At this phase, pairing of homologous chromosomes of maternal and paternal origin is initiated. Homologous pairing can be described as a process in which chromosomes actively search for each other. After finding their mates, they align themselves side by side with a slight space separating them.


Histology_Ross

Histology_Ross.  Zygotene. Synapsis, the close association of homologous chromosomes, begins at this stage and continues throughout pachytene. This process involves the formation of a synaptonemal complex, a tripartite structure that binds the chromosomes together. The synaptonemal complex is often compared to railroad tracks with an additional third rail positioned in the middle between two others. The cross ties in this track are represented by the transverse filaments that bind the scaffold material of both homologous chromosomes together.  Pachytene. At this stage, synapsis is complete. Crossing-over occurs early in this phase and involves transposition of DNA strands between two different chromosomes.


Histology_Ross

Histology_Ross.  Pachytene. At this stage, synapsis is complete. Crossing-over occurs early in this phase and involves transposition of DNA strands between two different chromosomes.  Diplotene. Early in this stage, the synaptonemal complex dissolves, and the chromosomes condense further. Homologous chromosomes begin to separate from each other and appear to be connected by newly formed junctions between chromosomes called chiasmata (sing., chiasma). Sister chromatids still remain closely associated with each other. Chiasmata indicate that crossing-over may have occurred.  Diakinesis. The homologous chromosomes condense and shorten to reach their maximum thickness, the nucleolus disappears, and the nuclear envelope disintegrates.


Histology_Ross

Histology_Ross.  Diakinesis. The homologous chromosomes condense and shorten to reach their maximum thickness, the nucleolus disappears, and the nuclear envelope disintegrates. Metaphase I is similar to the metaphase of mitosis except that the paired chromosomes are aligned at the equatorial plate with one member on either side. The homologous chromosomes are still held together by chiasmata. At late metaphase, chiasmata are cleaved and the chromosomes separate. Once the nuclear envelope has broken down, the spindle micro-tubules begin to interact with the chromosomes through the multilayered protein structure, the kinetochore, which is usually positioned near the centromere (see Fig. 3.13). The chromosomes undergo movement to ultimately align their centromeres along the equator of the spindle.


Histology_Ross

Histology_Ross. Anaphase I and telophase I are similar to the same phases in mitosis except that the centromeres do not split. The sister chromatids, held together by cohesin complexes and by the centromere, remain together. A maternal or paternal member of each homologous pair, now containing exchanged segments, moves to each pole. Segregation or random assortment occurs because the maternal and paternal chromosomes of each pair are randomly aligned on one side or the other of the metaphase plate, thus contributing to genetic diversity. At the completion of meiosis I, the cytoplasm divides. Each resulting daughter cell (a secondary spermatocyte or oocyte) is haploid in chromosome number (1n) and contains one member of each homologous chromosome pair. The cell is still diploid in DNA content (2d).


Histology_Ross

Histology_Ross. After meiosis I, the cells quickly enter meiosis II without passing through an S phase. Meiosis II is an equatorial division and resembles mitosis. During this phase, the proteinase enzyme separase cleaves the cohesion complexes between the sister chromatids. Cleavage of the cohesin complexes in the region of the centromere releases the bond between both centromeres. This cleavage allows the sister chromatids to separate at anaphase II and move to opposite poles of the cell. During meiosis II, the cells pass through prophase II, metaphase II, anaphase II, and telophase II. These stages are essentially the same as those in mitosis except that they involve a haploid set of chromosomes (1n) and produce daughter cells that have only haploid DNA content (1d). Unlike the cells produced by mitosis, which are genetically identical to the parent cell, the cells produced by meiosis are genetically unique.


Histology_Ross

Histology_Ross. In humans, as in all other multicellular organisms, the rates of cell proliferation and cell death determine the net cell production. An abnormality in any of these rates can cause disorders of cell accumulation (e.g., hyperplasia, cancer, autoimmune diseases) or disorders of cell loss (atrophy, degenerative diseases, AIDS, ischemic injury). Therefore, the balance (homeostasis) between cell production and cell death must be carefully maintained (Fig. 3.17). Cell death may occur as a result of acute cell injury or an internally encoded suicide program. Cell death may result from accidental cell injury or mechanisms that cause cells to self-destruct. The major two different mechanisms of cell death are necrosis and apoptosis.


Histology_Ross

Histology_Ross. Cell death may result from accidental cell injury or mechanisms that cause cells to self-destruct. The major two different mechanisms of cell death are necrosis and apoptosis.  Necrosis, or accidental cell death, is a pathologic process. It occurs when cells are exposed to an unfavorable physical or chemical environment (e.g., hypothermia, hypoxia, radiation, low pH, cell trauma) that causes acute cellular injury and damage to the plasma membrane. Under physiologic conditions, damage to the plasma membrane may also be initiated by viruses, or proteins called perforins. Rapid cell swelling and lysis are two characteristic features of this process.


Histology_Ross

Histology_Ross.  Apoptosis [Gr., falling off, as petals from flowers] was referred to in the past as programmed cell death. Today the term programmed cell death is applied more broadly to any kind of cell death mediated by an intracellular death program, irrespective of the trigger mechanism. Apoptosis represents a physiologic process. During apoptosis, cells that are no longer needed are eliminated from the organism. This process may occur during normal embryologic development or other normal physiologic processes, such as follicular atresia in the ovaries. Cells can initiate their own death through activation of an internally encoded suicide program. Apoptosis is characterized by controlled autodigestion, which maintains cell membrane integrity; thus, the cell “dies with dignity” without spilling its contents and damaging its neighbors.


Histology_Ross

Histology_Ross. FIGURE 3.17 • Schematic diagram showing the relationship between cell death and cell division. Under normal physiologic conditions (homeostasis), the rates of cell division and cell death are similar. If the rate of cell death is higher than that of cell division, then a net loss of cell number will occur. Such conditions are categorized as cell loss disorders. When the situation is reversed and the rate of cell division is higher than the rate of cell death, then the net gain in cell number will be prominent, leading to a variety of disorders of cell accumulation. TABLE Overview of Characteristic Features Distinguishing Necrosis from Apoptosis 3.2 Features of Dying Cells Necrosis Apoptosis Cell swelling —Cell shrinkage —Damage to the plasma membrane —Plasma membrane blebbing —Aggregation of chromatin —Fragmentation of the nucleus —Oligonucleosomal DNA fragmentation —Random DNA degradation —Caspase cascade activation —


Histology_Ross

Histology_Ross. In addition, certain cells or their secretions found in the immune system are toxic to other cells (e.g., cytotoxic T lymphocytes, natural killer [NK] cells); they initiate processes that destroy designated cells (e.g., cancer-transformed or virus-infected cells). In contrast to necrosis and apoptosis, cytotoxic death does not involve one specifc mechanism. For example, cell death mediated by cytotoxic T lymphocytes combines some aspects of both necrosis and apoptosis. For an overview of the apoptosis and necrosis, see Table 3.2. Necrosis begins with impairment of the cell’s ability to maintain homeostasis.


Histology_Ross

Histology_Ross. Necrosis begins with impairment of the cell’s ability to maintain homeostasis. As a result of cell injury, damage to the cell membrane leads to an influx of water and extracellular ions. Intracellular organelles such as the mitochondria, rER, and nucleus undergo irreversible changes that are caused by cell swelling and cell membrane rupture (cell lysis). As a result of the ultimate breakdown of the plasma membrane, the cytoplasmic contents, including lysosomal enzymes, are released into the extracellular space. Therefore, necrotic cell death is often associated with extensive surrounding tissue damage and an intense infammatory response (Fig. 3.18). Apoptosis is a mode of cell death that occurs under normal physiologic conditions.


Histology_Ross

Histology_Ross. Apoptosis is a mode of cell death that occurs under normal physiologic conditions. In apoptosis, the cell is an active participant in its own demise (“cellular suicide”). This process is activated by a variety of extrinsic and intrinsic signals. Cells undergoing apoptosis show the following characteristic morphologic and biochemical features (see Fig. 3.18);  DNA fragmentation occurs in the nucleus and is an irre versible event that commits the cell to die. DNA fragmen tation is a result of Ca2-dependent and Mg2-dependent activation of nuclear endonucleases. These enzymes selec tively cleave DNA, generating small oligonucleosomal fragments. Nuclear chromatin then aggregates, and the nucleus may divide into several discrete fragments bounded by the nuclear envelope.


Histology_Ross

Histology_Ross.  Decrease in cell volume is achieved by shrinking of the cytoplasm. The cytoskeletal elements become reorganized in bundles parallel to the cell surface. Ribosomes become clumped within the cytoplasm, the rER forms a series of concentric whorls, and most of the endocytotic vesicles fuse with the plasma membrane.


Histology_Ross

Histology_Ross.  Loss of mitochondrial function is caused by changes in the permeability of the mitochondrial membrane channels. The integrity of the mitochondrion is breached, the mitochondrial transmembrane potential drops, and the electron-transport chain is disrupted. Proteins from the mitochondrial intermembrane space, such as cytochrome c, are released into the cytoplasm to activate a cascade of proteolytic enzymes called caspases that are responsible for dismantling the cell. The regulated release of cytochrome c suggests that mitochondria, under the influence of Bcl-2 proteins (see page 95), are the decision makers for initiating apoptosis. Thus, many researchers view mitochondria either as the “headquarters for the leader of a crack suicide squad” or as a “high-security prison for the leaders of a military coup.”  Membrane blebbing results from cell membrane alterations. One alteration is related to translocation of certain molecules (e.g., phosphatidylserine) from the cytoplasmic surface


Histology_Ross

Histology_Ross. a military coup.”  Membrane blebbing results from cell membrane alterations. One alteration is related to translocation of certain molecules (e.g., phosphatidylserine) from the cytoplasmic surface to the outer surface of the plasma membrane. These changes cause the plasma membrane to change its physical and chemical properties and lead to blebbing without loss of membrane integrity (see Fig. 3.18).


Histology_Ross

Histology_Ross.  Formation of apoptotic bodies, the final step of apoptosis, results in cell breakage (Fig. 3.19a, b, and c). These membrane-bounded vesicles originate from the cytoplasmic bleb containing organelles and nuclear material. They are rapidly removed without a trace by phagocytotic cells. The removal of apoptotic bodies is so efficient that no inflammatory response is elicited. Apoptosis occurs more than 20 times faster than mitosis; therefore, it is challenging to find apoptotic cells in a routine H&E preparation (Fig. 3.19d). Apoptosis is regulated by external and internal stimuli.


Histology_Ross

Histology_Ross. Apoptotic processes can be activated by a variety of external and internal stimuli. Some factors, such as tumor necrosis factor (TNF), acting on cell membrane receptors, trigger apoptosis by recruiting and activating the caspase cascade. Consequently, the TNF receptor is known as the “death receptor.” Other external activators of apoptosis include transforming growth factor (TGF-), certain neurotransmitters, free radicals, oxidants, and UV and ionizing radiation. Internal activators of apoptosis include oncogenes (e.g., myc and rel ), tumor suppressors such as p53, and nutrient-deprivation antimetabolites (Fig. 3.20). Apoptotic pathways are also activated by the events leading to mitotic catastrophe—namely, malfunction of specific DNA-damage checkpoints in the cell cycle (see page 87). Mitotic catastrophe is accompanied by chromatin condensation, mitochondrial release of cytochrome c, activation of the caspase cascade, and DNA fragmentation.


Histology_Ross

Histology_Ross. Apoptosis can also be inhibited by signals from other cells and the surrounding environment via so-called survival factors. These include growth factors, hormones such as estrogen and androgens, neutral amino acids, zinc, and interactions with extracellular matrix proteins. Several cellular and viral proteins act as caspase inhibitors; for instance, nerve cells contain neuronal apoptosis inhibitory protein (NAIP) to protect them from premature apoptosis. However, the most important regulatory function in apoptosis is ascribed to internal signals from the Bcl-2 family of proteins. Members of this family consist of antiapoptotic and proapoptotic members that determine the life or death of a cell. These proteins interact with each other to suppress or propagate their own activity by acting on the downstream activation of various executional steps of apoptosis. They also act independently on mitochondria to regulate the release of cytochrome c, the most potent apoptosis-inducing agent.


Histology_Ross

Histology_Ross. Other Forms of Programmed Cell Death Several forms of programmed cell death were recently identified that differ from apoptosis or necrosis. There are several identified different forms of programmed cell death that do not fit into the classical apoptosis or necrosis scheme. They include the following.


Histology_Ross

Histology_Ross. There are several identified different forms of programmed cell death that do not fit into the classical apoptosis or necrosis scheme. They include the following. FIGURE 3.18 • Schematic diagram of changes occurring in necrosis and apoptosis. This diagram shows the major steps in necrosis and apoptosis. In necrosis (left side), breakdown of the cell membrane results in an influx of water and extracellular ions, causing the organelles to undergo irreversible changes. Lysosomal enzymes are released into the extracellular space, causing damage to neighboring tissue and an intense inflammatory response. In apoptosis (right side), the cell shows characteristic morphologic and biochemical features such as DNA fragmentation, decrease in cell volume, membrane blebbing without loss of membrane integrity, and formation of apoptotic bodies, causing cell breakage. Apoptotic bodies are later removed by phagocytotic cells without inflammatory reactions.


Histology_Ross

Histology_Ross.  Autophagy is a regulated cellular process that enables cells to turn over their contents by lysosomal degradation of their own components. It starts when an intracellular membrane (often part of sER cistern) wraps around an or-ganelle or portion of cytoplasm, forming a closed double membrane–bound vacuole. This vacuole, called an


Histology_Ross

Histology_Ross. FIGURE 3.19 • Electron micrographs of apoptotic cells. a. This electron micrograph shows an early stage of apoptosis in a lymphocyte. The nucleus is already fragmented, and the irreversible process of DNA fragmentation is turned on. Note the regions containing condensed heterochromatin adjacent to the nuclear envelope. 5,200. b. Further fragmentation of DNA. The heterochromatin in one of the nuclear fragments (left) begins to bud outward through the envelope, initiating a new round of nuclear fragmentation. Note the reorganization of the cytoplasm and budding of the cytoplasm to produce apoptotic bodies. 5,200. c. Apoptotic bodies containing fragments of the nucleus, organelles, and cytoplasm. These bodies will eventually be phagocytosed by cells from the mononuclear phagocytotic system. 5,200. (Courtesy of Dr. Scott H. Kaufmann, Mayo Clinic.) d. This photomicrograph taken with light microscopy of intestinal epithelium from the human colon shows apoptotic bodies (AB) within a single


Histology_Ross

Histology_Ross. 5,200. (Courtesy of Dr. Scott H. Kaufmann, Mayo Clinic.) d. This photomicrograph taken with light microscopy of intestinal epithelium from the human colon shows apoptotic bodies (AB) within a single layer of absorptive cells. BM, basement membrane. 750.


Histology_Ross

Histology_Ross. autophagosome, initially devoid of any lysosomal enzymes, fuses with lysosomes and initiates digestion. For a detailed description of three pathways utilized in autophagy, see pages 43 to 44.  Mitotic catastrophe is a type of cell death that occurs during mitosis. It results from a combination of cellular damage and malfunction of several cell-cycle checkpoints such as the G1, S, and G2 DNA-damage checkpoints or the spindle-assembly checkpoint (page 87). Failure to arrest the cell cycle before mitosis occurs causes problems with chromosome separation, which triggers the apoptotic pathway and cell death.