extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Mammalian Thioredoxin Reductase Is Irreversibly Inhibited by Dinitrohalobenzenes by Alkylation of Both the Redox Active Selenocysteine and Its Neighboring Cysteine Residue*

The immunostimulatory dinitrohalobenzene compound 1-chloro-2,4-dinitrobenzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase (TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in the modified enzyme (Arnér, E. S. J., Björnstedt, M., and Holmgren, A. (1995) J. Biol. Chem. 270, 3479–3482). Here we have further analyzed the reactivity with the enzyme of DNCB and analogues with varying immunomodulatory properties. We have also identified the reactive residues in bovine thioredoxin reductase, recently discovered to be a selenoprotein. We found that 4-vinylpyridine competed with DNCB for inactivation of TrxR, with DNCB being about 10 times more efficient, and only alkylation with DNCB but not with 4-vinylpyridine induced an NADPH oxidase activity. A number of nonsensitizing DNCB analogues neither inactivated the enzyme nor induced any NADPH oxidase activity. The NADPH oxidase activity of TrxR induced by dinitrohalobenzenes generated superoxide, as detected by reaction with epinephrine (the adrenochrome method). Addition of superoxide dismutase quenched this reaction and also stimulated the NADPH oxidase activity. By peptide analysis using mass spectrometry and Edman degradation, both the cysteine and the selenocysteine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where Sec indicates selenocysteine) were determined to be dinitrophenyl-alkylated upon incubation of native TrxR with NADPH and DNCB. A model for the interaction between TrxR and dinitrohalobenzenes is proposed, involving a functional FAD in the alkylated TrxR generating an anion nitroradical in a dinitrophenyl group, which in turn reacts with oxygen to generate superoxide. Production of reactive oxygen species and inhibited reduction of thioredoxin by the modified thioredoxin reductase after reaction with dinitrohalobenzenes may play a major role in the inflammatory reactions provoked by these compounds.

The immunostimulatory dinitrohalobenzene compound 1-chloro-2,4-dinitrobenzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase (TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in the modified enzyme (Arné r, E. S. J., Bjö rnstedt, M., and Holmgren, A. (1995) J. Biol. Chem. 270, 3479 -3482). Here we have further analyzed the reactivity with the enzyme of DNCB and analogues with varying immunomodulatory properties. We have also identified the reactive residues in bovine thioredoxin reductase, recently discovered to be a selenoprotein. We found that 4-vinylpyridine competed with DNCB for inactivation of TrxR, with DNCB being about 10 times more efficient, and only alkylation with DNCB but not with 4-vinylpyridine induced an NADPH oxidase activity. A number of nonsensitizing DNCB analogues neither inactivated the enzyme nor induced any NADPH oxidase activity. The NADPH oxidase activity of TrxR induced by dinitrohalobenzenes generated superoxide, as detected by reaction with epinephrine (the adrenochrome method). Addition of superoxide dismutase quenched this reaction and also stimulated the NADPH oxidase activity. By peptide analysis using mass spectrometry and Edman degradation, both the cysteine and the selenocysteine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where Sec indicates selenocysteine) were determined to be dinitrophenyl-alkylated upon incubation of native TrxR with NADPH and DNCB. A model for the interaction between TrxR and dinitrohalobenzenes is proposed, involving a functional FAD in the alkylated TrxR generating an anion nitroradical in a dinitrophenyl group, which in turn reacts with oxygen to generate superoxide. Production of reactive oxygen species and inhibited reduction of thioredoxin by the modified thioredoxin reductase after reaction with dinitrohalobenzenes may play a major role in the inflammatory reactions provoked by these compounds.
Thioredoxin reductase from Escherichia coli has been extensively characterized (1), and a high resolution x-ray structure shows surprisingly large differences to the other members of the pyridine nucleotide-disulfide oxidoreductase family (8,9). Thus, the subunits of about 35 kDa are smaller than the about 50-kDa subunits present in glutathione reductase from all species. Furthermore the active site cysteine residues of E. coli TrxR are located in the central NADPH domain and separated by two amino acids (Cys-Ala-Thr-Cys), in comparison with the active site in glutathione reductase which is Cys-Val-Asn-Val-Gly-Cys and located in the NH 2 -terminal FAD domain, suggesting convergent evolution (9). The structural features of TrxR from E. coli with a high specificity for its homologous Trx are also typical for TrxR from prokaryotes, lower eukaryotes like yeast, or plants (1, 8 -10).
It has long been known that mammalian TrxR has properties strikingly different to the enzyme from E. coli and lower organisms (2)(3)(4). The enzymes from calf liver and thymus and rat liver were first purified to homogeneity and showed 58-kDa subunits (11,12). The mammalian thioredoxin reductases, including that of human placenta (13), are thereby larger than the E. coli enzyme and in contrast have a wide substrate specificity. Thus, the mammalian enzymes will reduce thioredoxins from different species (11), several low molecular weight disulfide substrates including DTNB used in assays (11,12) or lipoic acid (14) as well as other nondisulfide substrates including selenodiglutathione (15), selenite (16), alloxan (17), or most surprising lipid hydroperoxides (18). The wide substrate specificity indicates an unusual structure of the active site, which is also demonstrated by the inhibition of mammalian TrxR by several drugs in clinical use including antitumor quinones (19,20), nitrosoureas (21), or 13-cis-retinoic acid (22).
Recently, two proteins from transformed human cells having thioredoxin reductase activity, a 55-kDa subunit enzyme from a Jurkat T-cell line (23) and a 57-kDa subunit protein from a lung adenocarcinoma cell line (24), were shown to contain selenocysteine. A peptide sequence from the protein purified from the human T-cell line (23) agreed with a putative human placental TrxR cDNA sequence (25).
We have confirmed the sequence of the human enzyme and determined that of bovine TrxR peptides and a cDNA clone of rat TrxR (26). We have also shown that the selenocysteine residue, conserved between the mammalian species in the carboxyl-terminal motif Gly-Cys-Sec-Gly, is essential for catalytic activity (26). The mammalian TrxR amino acid sequence is highly homologous to GR and carries the identical sequence motif within the NH 2 -terminal FAD domain that contains the redox active cysteine residues of GR. The carboxyl-terminal motif with a penultimate Sec and a neighboring Cys residue is an elongation to the structure that is not present in GR, but is homologous to a Cys-Cys-containing elongation found in another related flavoprotein, mercuric reductase (26). The domain organization of TrxR is schematically depicted in Fig. 1.
DNCB is an electrophilic compound used as a substrate in assays to determine glutathione S-transferase, which is involved in elimination of DNCB in vivo (27). DNCB is therefore also used in cell culture experiments as a GSH-depleting agent (28). Furthermore, DNCB has an established use as an immunomodulatory agent to provoke delayed-type hypersensitivity (29). Although proposed to function as a hapten, the mechanism of DNCB immunomodulation is not clear (30).
To summarize our earlier findings, we showed that DNCB irreversibly inhibited mammalian TrxR with second order kinetics by alkylating the enzyme, but that TrxR had to be reduced by preincubation with NADPH for the alkylation to occur (31). Upon alkylation, an NADPH oxidase activity could be detected, which was about 30-fold increased as compared with that of the native enzyme, and no consumption of NADPH was seen under anaerobic conditions (31). In the present study we demonstrate that the nitro groups of the dinitrophenylalkylated TrxR are necessary for the induction of the NADPH oxidase activity, and we identify the residues alkylated by DNCB.

EXPERIMENTAL PROCEDURES
Materials and Enzymes-DNCB, DNFB, DNCB analogues, IAA, and 4-VP as well as epinephrine bitartrate salt and SOD were purchased from Sigma. Mammalian TrxR was purified from human placenta and calf thymus as described (12). Enzyme concentration was determined from A 280 nm measurements (A 280 Ϫ A 340 ϭ 0.7 mg/ml) or by the activity with 5 mM DTNB assuming that 1200 A 412 units/ml corresponded to 1 mg of protein/ml and an M r of 120,000, i.e, a specific activity of 22 mol of NADPH oxidized per min/mg of protein in the DTNB assay (12).
Enzyme Activity Measurements-All experiments were performed in 0.5 ml of 50 mM Tris-Cl, 2 mM EDTA, pH 8.0, at 20°C using a Zeiss PM Q3 or a Hitachi spectrophotometer with semimicro quartz cuvettes. Details of activity determination using selenite reduction have been given in a previous paper (16). DNCB or analogues were used as stock solutions in 99.5% EtOH, and in all samples the same final volume of EtOH (1%) was added, also to controls. For analysis of inhibition and induction of NADPH oxidase activity, 40 -150 nM TrxR was used with 100 M amounts of the dinitrobenzenes or DNCB analogues, 50 M IAA or 1 mM 4-VP. For determination of NADPH oxidase activity, oxidation of NADPH was followed at 340 nm using a molar extinction coefficient of 6200 M Ϫ1 cm Ϫ1 in calculations.
Isolation of dnp-alkylated TrxR with Sephadex G-25 Columns-TrxR was incubated with 200 M NADPH in 0.50 ml of 50 mM Tris-Cl, 2 mM EDTA, pH 8.0, containing 1 mg/ml bovine serum albumin. After incubation for 20 -60 min with electrophilic compounds at 20°C, each sample was applied to a Sephadex G-25 column (NAP-5, Amersham Pharmacia Biotech), equilibrated with 50 mM Tris-Cl, 2 mM EDTA, pH 8.0, and the enzyme was collected in 1.0 ml of buffer. The alkylated TrxR free from electrophilic compound in solution could then be further analyzed for enzyme activity. Nonalkylated TrxR treated in the same manner was used as control enzyme. Detection of Superoxide by the Adrenochrome Method-Superoxide was followed by reaction with epinephrine (2 mM in both reference and sample cuvettes) under production of adrenochrome, with an increase of absorbance at 480 nm using an extinction coefficient of 4020 M Ϫ1 cm Ϫ1 (32).

Reactivity of Dinitrohalobenzenes and Analogues with TrxR
and Production of Superoxide-Induction of an NADPH oxidase activity in mammalian TrxR upon alkylation with DNCB could be an effect due to the alkylation of the enzyme per se, due to specific properties of DNCB, or due to a combination of these factors. To analyze this, we compared the effects on mammalian TrxR of different DNCB analogues and of alkylating agents other than dinitrohalobenzenes. The well known alkylating agents IAA or 4-VP did not induce any NADPH oxidase activity, although TrxR was irreversibly inhibited, showing that only alkylation of the reduced enzyme was not sufficient to induce an NADPH oxidase activity (Table I). The DNCB analogues 3,4-DCNB, 2,5-DCNB, 4-chloronitrobenzene, and 1,4dichlorobenzene neither inhibited TrxR nor induced an NADPH oxidase activity, whereas a second dinitrohalobenzene compound, DNFB, both irreversibly inhibited TrxR and induced an NADPH oxidase activity (Table I). The results indicated that specific properties of dinitrohalobenzenes were needed to concomitantly alkylate and induce an NADPH oxi-  (26). Mammalian TrxR has close to 500 amino acid residues, and the active enzyme is a homodimer. The FAD and NADPH binding domains as well as the central and interface domains indicated in the figure are suggested by the high homology to glutathione reductase (1,26). The sequences in the NH 2 -terminal FAD domain encompassing the active site disulfide motif identical to that of GR and the carboxyl-terminal elongation with the penultimate Sec and its neighboring Cys are shown by one-letter amino acid abbreviations. These motifs are conserved between the mammalian species (26). dase activity in TrxR.
Simultaneous incubation of TrxR with DNCB and 4-VP showed that the two compounds competed for the same site of alkylation. We found that, under the conditions utilized, a 5-10-fold excess of 4-VP decreased the DNCB alkylation of TrxR by about 50%, as illustrated by a 50% lower induction of NADPH oxidase activity at this ratio of 4-VP to DNCB (Fig. 2). After incubation with 5 M DNCB and 900 M 4-VP (a ratio of 180:1), almost all of the induction of an NADPH oxidase activity was blocked. It should be emphasized that in all samples shown in Fig. 2, TrxR was completely inactivated for its normal catalytic reactions, illustrating that a complete alkylation of the enzyme had occurred in all of the samples, but that only alkylation with DNCB induced the NADPH oxidase activity.
We noted that high concentrations of DNCB seemed to further increase the NADPH oxidase activity of TrxR, indicating that also DNCB in solution could induce an NADPH oxidase activity. To further study this, TrxR was alkylated with DNCB, then desalted and analyzed for NADPH oxidase activity with addition of increasing concentrations of DNCB. This confirmed that free DNCB in solution could lead to an NADPH oxidase activity, and this effect showed classic Michaeli-Menten type kinetics (Fig. 3). The K m for DNCB in this reaction was 0.21 mM and the maximal NADPH turnover dependent on DNCB in solution was 17 min Ϫ1 . The induced NADPH oxidase activity of TrxR alkylated by DNCB but desalted and with no additional DNCB present in the solution was 4.5 min Ϫ1 . Thereby the total maximal DNCB-dependent NADPH oxidase activity of TrxR (i.e. the sum of the inherent activity of the alkylated enzyme and that dependent on DNCB in solution) was 22 min Ϫ1 (Fig. 3).
To confirm the possible formation of superoxide in the NADPH oxidase reaction, adrenochrome formation from epinephrine (32) was followed simultaneously with detection of oxidation of NADPH, and the effects of SOD on this system were analyzed (Table II). It was clear that adrenochrome was formed during the reaction and that SOD could totally inhibit this formation, demonstrating that superoxide indeed was    formed. The NADPH oxidation also increased when SOD was added, to twice the basal level when SOD had been added in amounts sufficient enough to dismute close to all of the superoxide formed, as indicated by the total inhibition of formation of adrenochrome (Table II). If hydrogen peroxide formed by the action of SOD or the decrease in levels of superoxide was the reason for the increased NADPH oxidation upon SOD addition is not clear and has to be investigated further.
Identification of Alkylated Residues-To determine the site(s) of alkylation, DNCB was in the presence of NADPH added to native TrxR at a 100-fold molar excess for 20 min at room temperature, i.e. conditions that yield a fully inactivated enzyme but concentrations and time of incubation sufficiently low to avoid extensive nonspecific alkylation. The dnp-alkylated protein was then desalted, digested with lysine-specific endoproteinase, and peptides were separated using reverse phase HPLC. Three fractions were found to exhibit absorbance at 340 nm, indicating the possible presence of dnp-alkylated peptides (Fig. 4, fractions A, B, and C). These fractions were subjected to Edman degradation, where fraction A gave no amino acid sequence, fraction B contained a peptide corresponding to the carboxyl-terminal end of the enzyme, and fraction C contained two peptides from the interface domain of the enzyme (Table III). When detection of phenylthiohydantoinderived amino acids was changed to 340 nm at the Edman degradation of the peptide in fraction B, a peak of decreasing intensity was seen during the first 2-3 cycles (not shown), which indicated that the dnp group is lost from the alkylated amino acid(s) during Edman degradation, as has been suggested elsewhere in experiments with glutathione S-transferase (33). Therefore Edman degradation could not be utilized to positively identify the residue(s) alkylated with a dnp group.
To determine residues alkylated in the peptides, we therefore utilized mass spectrometric analysis. The spectra of the analysis are given in Fig. 5 and summarized in Table IV. We found that fraction A, which gave no amino acid sequence, lacked higher molecular weight compounds that could correspond to alkylated peptides (Fig. 5A). However, the mass of compounds present in the two other fractions correlated well with the theoretical mass of dnp-alkylated peptide derivatives.
Fraction B contained the dnp-alkylated carboxyl-terminal peptide (Table III) and mass spectrometry (Fig. 5B) revealed the presence of three high molecular weight compounds (1397, 1644, and 3003 mass units, respectively). It should be noted that among the amino acid residues present in the carboxylterminal peptide, only the Cys or Sec residues could theoretically be alkylated by DNCB. The mass of none of the three compounds in the fraction correlated to a native form carboxylterminal peptide with only one dnp group attached. Instead, the compound with a mass of 1644 agreed well with the theoretical mass of the peptide alkylated at both the Cys and the Sec residues. The 1397 compound probably represented the product of a loss of Se(dnp) from the peptide with formation of dehydroalanine at the position of Sec. The 3003 compound could be proposed to represent a dimeric compound, linked by an oxygen bridge with the Sec residues in the form of selenenic acid. The structures of these proposed derivatives of the carboxyl-terminal peptide are shown in Fig. 6. The 1397 and 3003 compounds were most likely formed at drying of the sample or due to the process of mass spectrometry, while the doubly alkylated peptide should be the species originally present in fraction B at the stage of the HPLC separation.
Mass spectrometry of fraction C (Fig. 5C) showed that the peptide corresponding to the start of the interface region (peptide 1, Table III)  Peptides from dnp-alkylated TrxR produced with endoproteinase Lys-C were separated by reverse phase HPLC as described under "Experimental Procedures." Peptides were followed at 214 and 280 nm, and dnp-alkylated peptides were identified by absorbance at 340 nm. The fractions marked "A," "B," and "C" were subjected to further analysis using Edman degradation (Table III) or mass spectrometry (Fig. 5 and Table IV). a Fractions "A," "B," or "C" from Fig. 4. The results from mass spectrometric analysis of these fractions are given in Table IV and Fig. 5. b Determined by Edman degradation. Residues shown in italics were not unambiguously detected in the present study but have been determined previously (26). Underlined residues are those that theoretically could be alkylated with DNCB. For yields at the Edman degradation, see "Experimental Procedures." FIG. 5. Mass spectrometric analysis of fractions "A," "B," and "C" from the HPLC separation shown in Fig. 4. Electrospray mass spectra were recorded as described under "Experimental Procedures." Shown are the original spectra with the molecular weights of major singly charged ions given in parentheses and sodium adducts marked with asterisks. In the insets enlargements of relevant areas are given, with calculations of the parent compound molecular weights. A shows the analysis of fraction A from Fig. 4, B of fraction B, and C of fraction C. A summary of the analysis with an interpretation of compounds present is given in Table IV. peptide was not present in its nonalkylated form with a mass of 2575.3, but had been alkylated with one dnp group, representing the compound with a mass of 2744 (Table IV). DISCUSSION The NADPH oxidase activity of native TrxR was with 200 M NADPH determined to be about 0.15 min Ϫ1 (31), which is quite low compared with other flavoproteins (34). The increase of NADPH oxidase activity in mammalian TrxR by DNCB was shown here to be dependent not solely on the alkylation but on a combination of this with a certain property of the dinitrohalobenzenes, no doubt carried by the reactive nitro groups of these compounds.
The induced NADPH oxidase activity resembles that of GR incubated with TNBS studied in detail by Carlberg and Mannervik (35). This interaction was, however, reversible and was fully dependent on TNBS in solution. The kinetics of the TNBS induced NADPH oxidase activity in GR was most complex, sigmoidal in the absence and nonsigmoidal in the presence of NADP ϩ , with the latter supporting one-electron transfer reactions (35). However, with TrxR alkylated by DNCB, addition of NADP ϩ did not change the velocity of NADPH oxidase activity (not shown) and the effects of DNCB in solution added to DNCB-alkylated TrxR (Fig. 3) did not show the complex kinetics that was found in the interaction between GR and TNBS. Lipoamide dehydrogenase, which like GR is structurally related to mammalian TrxR (26), is known to carry diaphorase activity, i.e. having the capacity to transfer electrons to artificial acceptors such as dichlorophenolindophenol or ferricyanide (reviewed in Ref. 1). It is clear that the FAD of lipoamide dehydrogenase catalyzes this diaphorase activity directly without participation of the redox active disulfide, since both lipoamide dehydrogenase treated with -SH alkylators (36) point mutated lipoamide dehydrogenase with serines instead of cysteines at the redox active disulfide (37), or other chemical modifications inhibiting the normal redox reactions (1) can leave the electron transfer to alternate acceptors unaffected or even greatly increased.
The NAD(P)H-dependent aerobic redox cycling of aromatic nitro compounds with flavoenzymes under production of superoxide is a known phenomenon (36, 38 -40). The overall mechanism is believed to be reduction of the flavin by the pyridine nucleotide, followed by one-electron transfers from the flavin to the nitro groups under production of nitro anion radicals, that in turn react with molecular oxygen under production of superoxide. We believe that this is the basic mechanism for the NADPH oxidase activity in mammalian TrxR induced by dinitrohalobenzenes and that the reacting nitro groups are either in solution or, alternatively, those positioned close to the active site by a dinitrobenzene alkyl group of the alkylated enzyme.
We found that three residues were dnp-alkylated in TrxR upon gentle incubation with DNCB. The fraction with the highest absorbance at 340 nm (fraction B, Fig. 4) contained the peptide corresponding to the carboxyl-terminal end of the enzyme (see Table III and Fig. 1) with both the Cys and the Sec alkylated. In addition, we found a peptide corresponding to a part of the interface region being alkylated with one dnp group, either at the His proposed to act as a base at the active site in GR and TrxR-like enzymes (1,26) or at its neighboring Cys Loss of oxygen from dnp-alkylated peptide 2.
a Fraction "A," "B," and "C" from Fig. 4. b The mass spectrometry spectra are shown in Fig. 5. c The peptide derivatives suggested to have been present at stage of the HPLC shown in Fig. 4 are given in bold, while the other compounds are suggested to be artefacts arising from drying the fractions or the mass spectrometric analysis.  Table III (fraction B). Here are shown the structures of the three derivatives of this peptide proposed to be determined by mass spectrometric analysis of fraction B (Fig. 5B), as summarized in Table IV and the text. R denotes the 10 first amino acids of the peptide.
(peptide 2, fraction C, Table III). Alkylation at all three of these residues can in theory easily explain the inactivation of the normal enzymatic activity of the enzyme, all residues being envisioned to take part at the active site of the enzyme. It is not at this stage certain which of the nitro groups of the provided dnp groups that would give rise to superoxide formation. However, we may propose the following events. First, reduction of the enzyme with NADPH, via the FAD prosthetic group and probably the NH 2 -terminal GR-like redox active disulfide, must make the Cys and Sec at the carboxyl terminus, or possibly the whole active site, more accessible for alkylation, since oxidized TrxR is resistant to alkylation by DNCB (31) as well as digestion with carboxypeptidase Y (26). A combination of conformational change or reduction of a bridge between the carboxyl-terminal Cys and Sec residues 2 could be proposed to account for the change in accessibility for alkylation. We then propose that the FAD of the dnp-alkylated enzyme still can be reduced by NADPH but instead of two-electron transfer of reducing equivalents to the NH 2 -terminal active site disulfide, as in the normal case, a nitro group of the dnp group is reduced in a one-electron transfer to form an anion radical, which in turn reacts with oxygen to form superoxide. A second consecutive step of a one-electron transfer to form another nitro radical with subsequent reaction with oxygen under formation of superoxide would regenerate fully oxidized FAD of the enzyme and NO 2 groups in the participating dnp group. Adrenochrome formation is a good measure of superoxide formation formed by univalent reduction of oxygen by reduced flavins (32). The fact that 2 mol of adrenochrome were formed per mol of NADPH by the DNCB-alkylated TrxR and that SOD completely blocked this formation (Table II) supports the notion that two superoxides are formed per NADPH (or FADH 2 ). Free DNCB in solution is, in addition, proposed to be able to react with the FAD prosthetic group of the alkylated TrxR, to form nitroradicals and superoxide. This is supported by the Michaeli-Menten type of kinetics of the increase in NADPH oxidase activity dependent on free DNCB added to already alkylated TrxR (Fig. 3).
The cycle of events proposed above would explain the inactivation of TrxR with DNCB and concomitant induction of an NADPH oxidase activity. Since only three residues of the enzyme were found to be alkylated, the importance of these amino acids for the catalytic activity is illustrated. It should be pointed out that the active site structure of mammalian TrxR most likely is composed of the combination of the GR-like NH 2 -terminal redox active disulfide motif of one subunit and the carboxyl-terminal selenocysteine containing redox active motif of the other subunit (26), in analogy with mercuric reductase (1,41). This implies that the nitro groups of the dnp groups at the carboxyl-terminal motif could be proposed to become positioned in the vicinity of the NH 2 -terminal redox active motif, which normally contains the cysteine residues that accept reducing equivalents from FADH 2 .
In this study we could not detect dnp derivatization of the NH 2 -terminal GR-like active site dithiol/disulfide motif, which we previously found to be alkylated by 4-VP in a similar experiment with NADPH-dependent alkylation and identification of redox active residues of the enzyme (26). The explanation could be that, specifically, the nitro groups of DNCB sterically hinders alkylation of the NH 2 -terminal redox active motif, or, more likely, that the carboxyl-terminal motif generally is more accessible and easily alkylated. It should be noted that the conditions for alkylation in this study were milder than in that with 4-VP (26).
It is of interest to note that the Cys as well as the Sec residue of the carboxyl-terminal peptide had been alkylated. The selenol group of Sec usually has a high nucleophilic reactivity and a low pK a value, which makes this residue a natural target for alkylation with electrophilic compounds, providing that it is sterically accessible. The finding that the neighboring Cys residue was alkylated as well indicates that in mammalian TrxR, its sulfhydryl group has an unusual reactivity indicating a low pK a value.
Does the specific and high reactivity with mammalian TrxR of dinitrohalobenzenes like DNCB play a role in the mechanism of the immunomodulating properties of these compounds? In this context, it is of importance to note that all of the DNCB analogues that failed to inhibit TrxR or to induce any NADPH oxidase activity (Table I) previously had been tested in vivo for induction of hypersensitivity reactions and shown to provoke no reaction (30). In the same study, O 2 utilization, H 2 O 2 production, and NADPH consumption in skin or liver microsomes was also measured upon addition of dinitrohalobenzenes or the DNCB analogues. All of these properties correlated well to mouse ear swelling upon application of the compounds, whereas changes in levels of GSH or GSSG did not (30). The enzyme(s) responsible for the NADPH consumption and superoxide (or H 2 O 2 ) production were not identified, but based upon our study it is safe to conclude that TrxR is a strong candidate. How would the interaction with TrxR by dinitrohalobenzenes take part in the mechanism of immunostimulation by these compounds? Two mechanisms would be the most probable. First, Trx plays a central role in redox regulation of cell function (3,5,(42)(43)(44), and an irreversible inhibition of TrxR would therefore with certainty affect Trx-related functions in the immune system. Second, the induced NADPH oxidase activity and increased production of superoxide (and H 2 O 2 in the presence of SOD) by TrxR alkylated by dinitrohalobenzenes in combination with inhibition of the many anti-oxidant functions of the mammalian Trx system should give rise to a significant oxidative stress, which in itself is immunostimulatory (45).


extfieldsofstudy: Medicine Biology

text:
Regulation of αIIbβ3 Function in Human B Lymphocytes*

We studied the function of the platelet integrin αIIbβ3 using a B lymphocyte model in which αIIbβ3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate αIIbβ3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and αIIbβ3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Gαi, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of αIIbβ3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that αIIbβ3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Gαi, protein kinase C, and the actin cytoskeleton.

Exposure of a binding site for ligands such as fibrinogen and von Willebrand factor on the platelet integrin ␣IIb␤3 is a prerequisite for platelet aggregation (1). The process by which this binding site is exposed has been termed "inside-out" signaling and is initiated when agonist-generated intraplatelet signals induce a conformational change in ␣IIb␤3 by interacting with its cytoplasmic tails (2). To study the process of ␣IIb␤3 activation in vitro, we have developed a model system in which wild-type and mutant ␣IIb␤3 expressed in GM1500 cells, an Epstein-Barr virus-transformed human B lymphocyte line, can be induced to interact with soluble and immobilized fibrinogen by the phorbol ester phorbol 12-myristate 13-acetate (PMA) 1 (3,4). Using this system, we observed that the cytoplasmic tail of ␣IIb is not required for ␣IIb␤3 function in lymphocytes, that the conserved GFFKR motif in the ␣IIb tail is required for ␣IIb to interact with ␤3, and that signals interacting with the ␤3 cytoplasmic tail are responsible for the ability of agonists to stimulate ␣IIb␤3 function (4).
Phorbol esters such as PMA activate the conventional and novel isoforms of protein kinase C (PKC) (5). While phorbol esters are a potent stimulus for ligand binding to ␣IIb␤3 in platelets (6), they bypass the more proximal signaling events that are initiated when agonists bind to their cognate platelet membrane receptors. Most of the known platelet receptors for agonists are seven-transmembrane domain proteins that are coupled to various G proteins (7). Stimulation of these receptors on platelets is known to activate phospholipase C␤, generate diacylglycerol and inositol triphosphate, increase cytosolic calcium, and activate several isoforms of PKC (7). We were interested in determining whether stimulation of a G proteincoupled receptor on B lymphocytes would also expose the ligand-binding domain of ␣IIb␤3. Honda et al. (8) have reported that stimulation of the human N-formyl peptide chemoattractant receptor (fPR) in murine B lymphocytes induces the ␣4␤1mediated adherence of these cells to VCAM-1. The fPR is a seven-transmembrane domain protein coupled to the G protein G␣ i in leukocytes (9). Accordingly, we coexpressed the human fPR and ␣IIb␤3 in GM1500 cells and tested the ability of the transfected cells to interact with immobilized and soluble fibrinogen. We found that like PMA, the chemoattractant peptide formyl Met-Leu-Phe (fMLP), an fPR agonist, increased the avidity of ␣IIb␤3 for immobilized fibrinogen and its affinity for soluble fibrinogen. Moreover, using a number of metabolic inhibitors, we outlined an ␣IIb␤3 activation pathway involving the G-protein G i , PKC, and the actin cytoskeleton.

EXPERIMENTAL PROCEDURES
Reagents and Materials-The anti-FLAG monoclonal antibody (mAb) M1 and the fluorescent Ca 2ϩ indicator fluo-3 AM were obtained from IBI-Kodak and Molecular Probes, respectively. Immulon 2 flat bottom microtiter plates were purchased from Dynatech Laboratories. PMA, fMLP, pertussis toxin (PTX), bovine serum albumin, ionomycin, Arg-Gly-Asp-Ser (RGDS), and cytochalasin D were purchased from Sigma. Human fibrinogen was obtained from Enzyme Research Labs. Bisindolylmaleimide I (BIM I), bisindolylmaleimide V (BIM V), and genistein were purchased from Calbiochem. Recombinant C3 exoenzyme was purchased from Upstate Biotechnology. Lipofectin reagent and Opti-MEM media were obtained from Life Technologies, Inc. G418 was purchased from Mediatech. Piceatannol and hygromycin were obtained from Boehringer Mannheim.
Coexpression of the Human fPR and ␣IIb␤3 in Human B Lymphocytes-Because GM1500 cells express ␤3, introduction of a cDNA for ␣IIb results in the expression of ␣IIb␤3 on the cell surface (4). Therefore, to coexpress the fPR and ␣IIb␤3 in GM1500 cells, a cDNA for ␣IIb in the plasmid pREP4 containing a gene for resistance to the antibiotic hygromycin (3) and a FLAG octapeptide-tagged cDNA for the human fPR in the plasmid pRc/CMV containing a gene for resistance to the antibiotic neomycin (a gift of Drs. James J. Campbell and Eugene Butcher, Stanford University) (10) were sequentially introduced into 7.5 ϫ 10 6 cells by electroporation (250 V and 960 microfarads). Stable co-transfectants were selected by growth in RPMI media containing 20% fetal calf serum and both G418 (750 g/ml) and hygromycin (200 g/ml). The simultaneous presence of ␣IIb␤3 and the fPR on the lymphocyte surface was confirmed by flow cytometry after staining the cells with either the ␣IIb␤3-specific mAb A2A9 (11) or the anti-FLAG mAb M1, followed by staining with fluorescein (FITC)-conjugated goat antimurine IgG. Flow cytometry was performed using a FACScan flow cytometer (Becton-Dickinson) as described previously (12).
To ensure that the expressed fPR was functional, 5 ϫ 10 7 cells were loaded with 5 M fluo-3 AM at room temperature for 30 min. The cells were then incubated with either 30 nM ionomycin or 100 nM fMLP for 30 s and Ca 2ϩ flux-induced fluorescence was measured with a FACScan flow cytometer as described previously (8).
Measurement of ␣IIb␤3 Function in Human B Lymphocytes-The ability of ␣IIb␤3 expressed by lymphocytes to interact with fibrinogen was tested by measuring agonist-stimulated lymphocyte adherence to immobilized fibrinogen (3) and agonist-stimulated binding of soluble FITC-fibrinogen using flow cytometry as described previously (4).
To measure lymphocyte adherence to fibrinogen, the wells of microtiter plates were coated with 10 g/ml purified human fibrinogen in 50 mM NaHCO 3 buffer, pH 8.0, containing 150 mM NaCl. Unoccupied protein-binding sites on the wells were blocked with 5 mg/ml bovine serum albumin dissolved in the same buffer. 1.5 ϫ 10 5 B lymphocytes, metabolically labeled overnight with [ 35 S]methionine, were suspended in 100 l of 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl 2 , 0.1% glucose, and 1% bovine serum albumin, stimulated with either PMA or fMLP, and added to the protein-coated wells. Following an incubation for 30 min at 37°C without agitation, the plates were vigorously washed four times with the suspension buffer and adherent cells were dissolved using 2% SDS. The SDS solutions were counted for 35 S in a liquid scintillation counter.
To measure the binding of soluble FITC-fibrinogen to agonist-stimulated lymphocytes, purified human fibrinogen (13) was labeled with FITC using a Calbiochem-FITC Labeling Kit as described by the manufacturer. Fibrinogen labeled with FITC in this manner remained monomeric as assessed by gel-filtration chromatography, supported platelet aggregation as well as unlabeled fibrinogen, and was 95% clottable with thrombin (13). 1.5 ϫ 10 5 B lymphocytes were then suspended in 100 l of 10 mM sodium phosphate buffer, pH 7.4, containing 137 mM NaCl, 1 mM CaCl 2 , and 1% bovine serum albumin (suspension buffer) and incubated with 0.25 M FITC-fibrinogen in the presence or absence of PMA or fMLP for 30 min at room temperature. The cells were washed once with suspension buffer and resuspended in a fixation solution consisting of 10 mM sodium phosphate buffer, pH 7.4, containing 137 mM NaCl and 0.37% formalin. Following a 10-min incubation on ice, the cells were again washed once with the suspension buffer, and analyzed by flow cytometry as described previously (4).
Effect of Botulinum C3 Exoenzyme on ␣IIb␤3 Function in GM1500 Cells-Recombinant C3 exoenzyme from Clostridium botulinum was introduced in GM1500 cells using Lipofectin Reagent. 1 ϫ 10 7 GM1500 cells were suspended in 1 ml of Opti-MEM media containing 40 g of Lipofectin and 12 g of C3 exoenzyme and incubated for 2 h at 37°C. The cell suspension was then divided into two aliquots. One aliquot was resuspended in complete media for 1 h and the ability of these cells to adhere to fibrinogen was tested as described above. ADP-ribosylation of the small GTPase RhoA during the incubation was examined using the second aliquot as described previously (14). Briefly, the cells were washed with Opti-MEM, resuspended in 50 l of 20 mM Tris-HCl buffer, pH 7.5, containing 0.25 M sucrose, 5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 2 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride, and homogenized by sonication. Following centrifugation of the homogenate at 1000 ϫ g for 5 min, 15 l of the supernatant were incubated with 50 ng of C3 exoenzyme and 10 M [ 32 P]NAD ϩ (Amersham) for 1 h at 30°C in 100 mM Tris-HCl buffer, pH 8.0, containing 20 mM nicotinamide, 10 mM thymidine, 10 mM dithiothreitol, and 5 mM MgCl 2 in a total reaction volume of 100 l. Sufficient SDS and dithiothreitol were added to make their final concentrations 3% and 200 mM, respectively, and the solution was heated at 100°C for 3 min. Following 0.1% SDS-10% polyacrylamide gel electrophoresis, ADP-ribosylated RhoA was visualized by autofluorography.

Introduction of a Functional fPR into GM1500 Cells-Previ-
ously, we demonstrated that the phorbol ester PMA induces the adherence of B lymphocytes expressing ␣IIb␤3 to immobilized fibrinogen (3). To determine whether adherence could also be induced by stimulating a receptor on the lymphocyte surface, we introduced plasmids containing a cDNA for the human fPR tagged at its 5Ј end with the FLAG epitope and a cDNA for ␣IIb into GM1500 B lymphocytes and selected for cells that stably expressed both proteins using the antibiotics G418 and hygromycin. As shown in Fig. 1A, the simultaneous presence of ␣IIb␤3 and the fPR on the lymphocyte surface was confirmed by flow cytometry after staining the cells with the ␣IIb␤3specific mAb A2A9 (11) and the anti-FLAG mAb M1. The cells also stained with a FITC-labeled derivative of fMLP (data not shown).
Measurement of Ca 2ϩ Flux in GM1500 Cells Expressing the fPR-fMLP induces a Ca 2ϩ flux in murine B lymphocytes expressing the human fPR (8). To verify that the fPR we expressed was functional, transfected GM1500 cells were loaded with the fluorescent Ca 2ϩ indicator fluo-3 AM and exposed to either 30 nM ionomycin or 100 nM fMLP. The resulting change in fluo-3 fluorescence was then measured by flow cytometry. As shown in Fig. 1B, ionomycin and fMLP induced comparable changes in fluo-3 fluorescence, confirming that fPR stimulation could induce a flux of Ca 2ϩ in the transfected cells (Fig. 1B). Moreover, the fMLP-stimulated Ca 2ϩ flux was inhibited by preincubating the cells for 2 h with 100 ng/ml PTX, indicating that the fPR in GM1500 cells is coupled to a PTX-sensitive G protein.
Comparison of PMA and fMLP-stimulated ␣IIb␤3 Function-Next, we compared the ability of fMLP and PMA to stimulate the adherence of lymphocytes expressing ␣IIb␤3 to immobilized fibrinogen. As shown in Fig. 2A, there was a concentration-dependent increase in lymphocyte adherence following exposure of the cells to fMLP. In multiple experiments, maximal adherence was observed at 300 nM fMLP, but was never more than one-half to two-thirds that induced by 200 ng/ml PMA. Moreover, no additive effect was seen when cells were stimulated simultaneously with fMLP and PMA, suggesting that these agonists were acting through the same signaling pathway.
The adherence of PMA-stimulated lymphocytes to immobilized fibrinogen is inhibited by soluble fibrinogen (4). As shown in Fig. 2B, the adherence of fMLP-stimulated cells also decreased as the concentration of soluble fibrinogen in the suspending buffer increased. Moreover, like cells stimulated by PMA (4), inhibition was maximal at a concentration of soluble fibrinogen of 40 -50 M.
Platelet stimulation induces a change in the affinity of ␣IIb␤3 for ligands and enables it to bind soluble fibrinogen (13). Previously, we found that PMA stimulation enabled transfected lymphocytes expressing ␣IIb␤3 to bind soluble fibrinogen (4). To determine if fPR stimulation would do likewise, we stimulated lymphocytes coexpressing ␣IIb␤3 and the fPR with either 200 ng/ml PMA or 300 nM fMLP and used flow cytometry to compare the binding of FITC-fibrinogen. As we had seen previously (4), the fluorescence histogram of PMA-stimulated lymphocytes was shifted substantially to the right of the histogram of unstimulated cells, indicating that fibrinogen was bound to the stimulated cells (Fig. 3A). The fluorescence histogram of fMLP-stimulated lymphocytes was also shifted to the right, although again fMLP was a less potent agonist than PMA (Fig. 3B). Preincubating both sets of stimulated cells with the mAb A2A9 prevented the shifts in fluorescence, confirming that the FITC-fibrinogen was bound to ␣IIb␤3. Moreover, like the binding of fibrinogen to stimulated platelets (13,15), fibrinogen binding to PMA and fMLP-stimulated lymphocytes reached saturation at a fibrinogen concentration of Ϸ200 g/ml and was abolished by either a 15-fold excess of unlabeled fibrinogen or 200 M RGDS (data not shown).
Effect of Signaling Pathway Inhibitors on ␣IIb␤3 Function in Lymphocytes-Because PTX inhibits fMLP-stimulated Ca 2ϩ flux in GM1500 cells expressing the fPR, we asked whether PTX would inhibit fMLP-stimulated ␣IIb␤3 function in these cells. As expected, preincubating the cells with PTX had no effect on PMA-stimulated adherence to fibrinogen (Fig. 4). However, PTX reduced fMLP-stimulated adherence to nearly baseline levels. On the other hand, no inhibition was observed when the cells were stimulated with both fMLP and PMA, indicating that PMA was able to bypass the PTX effect. Identical results were seen when FITC-fibrinogen binding, rather than cell adherence, was used as the indicator of ␣IIb␤3 function (data not shown).
The fMLP-and interleukin 8-stimulated adherence of murine B cells mediated by ␣4␤1 is unaffected by inhibiting PKC (16). To determine the effect of PKC inhibitors on fMLP-stimulated ␣IIb␤3 function in human B cells, we incubated transfected GM1500 cells overnight with nanomolar concentrations of either the high affinity PKC inhibitor BIM I (17) or the low affinity inhibitor BIM V (18) and measured agonist-stimulated lymphocyte adherence to fibrinogen. As expected, BIM I reduced PMA-stimulated adherence to baseline levels, whereas the same concentrations of BIM V had no effect (Fig. 5). However, to our surprise, BIM I, but not BIM V, also reduced fMLP-stimulated adherence to baseline levels. Identical results were seen when FITC-fibrinogen binding was measured instead of adherence (data not shown). Thus, these experiments suggest that fPR stimulation regulates ␣IIb␤3 function in human B cells via a signaling pathway that includes PKC.
Stimulation of integrin function is associated with the activation of a number of protein tyrosine kinases (19). To determine whether tyrosine phosphorylation regulates ␣IIb␤3 function in lymphocytes, we incubated transfected GM1500 cells overnight with micromolar concentrations of the tyrosine ki-nase inhibitor genistein (20) and measured agonist-stimulated lymphocyte adherence to fibrinogen. As shown in Fig. 6, genistein concentrations as high as 150 M had no effect on either PMA-or fMLP-stimulated lymphocyte adherence. A small degree of inhibition (29 and 16%, respectively) was observed at a genistein concentration of 300 M, a concentration at which genistein also affects the activity of serine/threonine kinases (20). Activation of the non-receptor tyrosine kinase Syk is an early event after platelet stimulation by agonists such as collagen and thrombin (21, 22), although in Epstein-Barr virustransformed lymphocytes, Syk activity is constitutively inhibited by the Epstein-Barr virus-encoded protein LMP2 (23). Nevertheless, to determine whether residual Syk activity in GM1500 cells could regulate ␣IIb␤3 function, we incubated transfected cells with the Syk inhibitor piceatannol (24) and measured both PMA and fMLP-stimulated lymphocyte adherence to fibrinogen. As shown in Fig. 6, 30 g/ml piceatannol, a concentration that completely inhibited collagen-induced platelet aggregation, had no effect on ␣IIb␤3 function in cells stimulated by either PMA or fMLP.
Role of the Actin Cytoskeleton in Regulating ␣IIb␤3 Function in Lymphocytes-Inhibitors of actin polymerization impair ␤1 and ␤2 integrin function in leukocytes, suggesting that the actin cytoskeleton regulates integrin function in these cells (14,16,25). Whether the actin cytoskeleton regulates ␣IIb␤3 function in platelets is less certain, although it was recently reported that the actin polymerization inhibitor cytochalasin E inhibits thrombin-stimulated ␣IIb␤3 function (26). To determine if the actin cytoskeleton regulates ␣IIb␤3 function in GM1500 cells, we measured the effect of cytochalasin D on unstimulated and PMA-stimulated lymphocyte adherence to fibrinogen. We found that increasing concentrations of cytocha- ␣IIb␤3 Function in B Lymphocytes lasin D inhibited PMA-stimulated adherence, with few adherent cells remaining at a cytochalasin D concentration of 10 M (Fig. 7). By contrast, submicromolar concentrations unexpectedly, but consistently, increased unstimulated adherence, such that adherence in the presence 0.01 M cytochalasin D was 2-3-fold greater than in its absence. We also examined the effect of cytochalasin D on FITC-fibrinogen binding to both PMA-stimulated and unstimulated GM1500 cells. As shown in Fig. 8A, 0.1 M cytochalasin D, but not 0.01 M cytochalasin D, completely inhibited FITC-fibrinogen binding to PMA-stimulated cells. Conversely, whereas cytochalasin D concentrations of 0.1 M or greater did not influence the interaction of FITCfibrinogen with unstimulated lymphocytes, 0.01 M cytochalasin D consistently induced FITC-fibrinogen binding to these cells, albeit to a limited degree (Fig. 8B).
The Rho family of small GTPases regulates a number of cellular functions such as shape, motility, and adhesion by reorganizing the actin cytoskeleton (27). In lymphocytes, inhibiting RhoA with C3 exoenzyme from C. botulinum impairs agonist-stimulated cell adhesion mediated by ␣L␤2 (14) and ␣4␤1 (16). C3 exoenzyme has also been reported to inhibit thrombin-induced platelet aggregation (28). To determine whether RhoA plays a role in the regulation of ␣IIb␤3 function in GM1500 cells, we introduced C3 exoenzyme into the cells using Lipofectin and measured its effect on PMA and fMLPstimulated adherence to fibrinogen. To verify that C3 exoenzyme had ADP-ribosylated RhoA in the Lipofectin-treated cells, an aliquot of these cells was homogenized and re-exposed to the enzyme in the presence of [ 32 P]NAD ϩ . As shown in Fig.  9A, there was a substantial reduction in the incorporation of 32 P into RhoA from cells that had been treated with Lipofectin in the presence of C3 exoenzyme compared with cells that had been treated with Lipofectin in its absence. This indicates that RhoA in the former cells had been ADP-ribosylated during the first incubation with C3 exoenzyme, rendering it resistant to ADP-ribosylation during the second. Nevertheless, as shown in Fig. 9B, inhibiting RhoA with C3 exoenzyme had essentially no effect on the ability of the cells to adherence to fibrinogen following stimulation with either low or high concentrations of PMA and fMLP. DISCUSSION To delineate signaling pathways that can convert ␣IIb␤3 from an inactive to a ligand binding conformation, we have expressed recombinant ␣IIb␤3 in human B lymphocytes. Initially, we found that the phorbol ester PMA not only induced the adherence of these cells to fibrinogen, but enabled ␣IIb␤3 to bind soluble fibrinogen (4). In the current work, we asked whether an extracellular agonist acting through its cognate membrane receptor could induce ␣IIb␤3 function in these cells. Stimulation of G protein-coupled receptors in platelets results in ␣IIb␤3 activation (7). Similarly, stimulation of these receptors leads to integrin activation in lymphocytes and other cells of the hematopoietic lineage (9). We found that by coexpressing ␣IIb␤3 with the human formyl peptide receptor, a seven-trans-membrane domain G protein-coupled receptor (9), we could induce B cell adherence to immobilized fibrinogen and soluble fibrinogen binding to ␣IIb␤3 using the chemoattractant peptide fMLP. Moreover, we found that the signaling pathway initiated by fPR stimulation involved PKC and similar to signaling initiated by PMA, resulted in a change in the actin cytoskeleton.
The human fPR is normally expressed in phagocytic cells where it transduces signals by activating the PTX-sensitive G proteins G␣ i2 or G␣ i3 (9). We found that PTX inhibited the fMLP-stimulated interaction of co-transfected GM1500 cells with both immobilized and soluble fibrinogen, implying that G␣ i activation can regulate ␣IIb␤3 function in these cells. Whether G␣ i can also regulate ␣IIb␤3 function in platelets is uncertain. However, PTX inhibits thrombin-stimulated phosphoinositide hydrolysis in saponin-permeabilized human platelets, suggesting that at least the thrombin receptor in human platelets can couple to G␣ i (29). On the other hand, platelets from G␣ q -deficient mice fail to aggregate in response to thrombin, ADP, collagen, arachidonic acid, and U46619, despite normal levels of G␣ i , indicating that PTX-insensitive G protein G␣ q couples agonist receptors to ␣IIb␤3 in murine platelets (30). Nonetheless, it is possible that the difference between human lymphocytes and murine platelets may simply reflect a difference in the types of signaling pathways that are present in human and murine cells, similar to a difference in the types

FIG. 4. Inhibition of PMA-and fMLP-stimulated lymphocyte adherence to fibrinogen by PTX.
To determine the effect of PTX on ␣IIb␤3-mediated lymphocyte adherence to fibrinogen, GM1500 cells coexpressing the fPR and ␣IIb␤3 were preincubated with either buffer or 100 ng/ml PTX for 2 h. Lymphocyte adherence to immobilized fibrinogen stimulated by 200 ng/ml PMA, 100 nM fMLP, or both agonists together was measured as described in the legend to Fig. 2 and under "Experimental Procedures." The data are expressed as the mean and S.E. of quadruplicate determinations.

FIG. 5. Inhibition of PMA-and fMLP-stimulated lymphocyte adherence to fibrinogen by the PKC inhibitors BIM I and BIM V.
The effect of BIM I (f) and BIM V (u) on ␣IIb␤3-mediated lymphocyte adherence to fibrinogen was determined by incubating GM1500 cells coexpressing the fPR and ␣IIb␤3 overnight with the indicated concentrations of the inhibitors. Lymphocyte adherence to immobilized fibrinogen was stimulated by either 200 ng/ml PMA or 100 nM fMLP and measured as described in the legend to Fig. 2 and under "Experimental Procedures." The data are expressed as the mean and S.E. of quadruplicate determinations.
FIG. 6. Effect of protein tyrosine kinase inhibitors on PMAand fMLP-stimulated lymphocyte adherence to fibrinogen. GM1500 cells coexpressing the fPR and ␣IIb␤3 were incubated overnight with either the protein tyrosine kinase inhibitor genistein at 150 M or the Syk inhibitor piceatannol at 30 g/ml. Lymphocyte adherence to immobilized fibrinogen was stimulated by either 200 ng/ml PMA (f) or 100 nM fMLP (u) and measured as described in the legend to of thrombin receptors expressed by human and murine platelets (31,32).
The ability of inhibitors of PKC (33) to inhibit platelet aggregation and/or ligand binding to ␣IIb␤3 implies that protein phosphorylation by PKC regulates ␣IIb␤3 activity. Moreover, the PKC activator PMA is a potent stimulus for ligand binding to ␣IIb␤3 on both human platelets (6) and G␣ q -deficient murine platelets (30). The identity of the proteins phosphorylated by PKC in platelets to regulate ␣IIb␤3 function is unknown. The ␤3 cytoplasmic tail has been found to contain phosphorylated ␣IIb␤3 Function in B Lymphocytes threonine residues after platelet stimulation by thrombin, PMA, or the thromboxane analogue U46619 (34). However, the fraction of ␤3 containing phosphorylated threonine in both resting and stimulated platelets was low and unlikely to affect the function of more than a few ␣IIb␤3 heterodimers. We found that the fMLP-stimulated interaction of ␣IIb␤3 with either immobilized or soluble fibrinogen in B cells was prevented by the specific PKC inhibitor BIM I. Thus, activation of PKC, either directly with PMA or via fPR activation of G␣ i , is sufficient to induce ␣IIb␤3 function in lymphocytes. On the other hand, Laudanna et al. (16) observed that inhibiting PKC had no effect on fMLP-stimulated ␣4␤1 function in murine B cells (16). Again, it is possible that this difference simply reflects differences between human and murine cells. It is also possible that at least two signaling pathways can be initiated by fPR stimulation and that these pathways can differentiate between ␤1 and ␤3 integrins. In support of this possibility, Weber et al. (35) found that signaling pathways arising from the receptors for the chemoattractants RANTES, MCP-3, and C5a in eosinophils can differentially regulate the function of ␣4␤1 and ␣L␤2. Whether PKC-independent pathways also exist in B cells that can regulate ␣IIb␤3 function remains to be determined.
Phosphorylation of tyrosines 747 and 759 in the ␤3 cytoplasmic tail has also been detected after thrombin-stimulated platelet aggregation (36). However, this phosphorylation was not observed after thrombin stimulation in the absence of aggregation, suggesting that it is a consequence of ligand binding to ␣IIb␤3 ("outside-in" signaling), rather than being part of the process of ␣IIb␤3 activation. Blystone et al. (37) also detected phosphorylation of ␤3 Tyr-747 after monocytes were exposed to Mn 2ϩ or platelets were treated with either Mn 2ϩ or thrombin. When they expressed ␣v␤3 heterologously in K562 cells, they found Tyr-747 phosphorylation necessary, but not sufficient, to support either PMA and thrombin-stimulated cell adhesion, and like Tyr-747 phosphorylation in platelets (36), required ligand binding to ␣v␤3. We found that the protein tyrosine kinase inhibitor genistein had no effect on the ability of ␣IIb␤3 in B lymphocytes to interact with fibrinogen. Similarly, we found that piceatannol, an inhibitor reportedly specific for the tyrosine kinase Syk found in lymphocytes and platelets (38), had no effect on either PMA-or fMLP-stimulated ␣IIb␤3 function in lymphocytes. Thus, our data, combined with the inability to detect significant amounts of phosphorylated serine or threonine on the ␤3 of stimulated platelets (34), suggest that phosphorylation of ␣IIb␤3 is not required to regulate its interaction with ligands.
The biochemical events that follow PKC activation in lymphocytes and platelets are uncertain. However, one consequence of PKC-mediated signaling is regulation of membranecytoskeletal interactions (39). For example, Kucik and coworkers (40) found that exposing human B lymphocytes to PMA increased the diffusion of ␣L␤2 in the plane of the lymphocyte membrane and augmented lymphocyte adherence to ICAM-1. Similar effects were observed following exposure of the lymphocytes to low concentrations of cytochalasin D. These data suggest that cytoskeletal constraints, released by either PKC activation or cytochalasin D, maintain ␣L␤2 in a low avidity state. Lub et al. (41) extended these observations by showing that maximum ␣L␤2-mediated lymphocyte adherence required both ␣L␤2 clustering and an increase in its affinity for ICAM-1. We found that micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of lymphocytes expressing ␣IIb␤3 with either immobilized or soluble fibrinogen. Conversely, we found that nanomolar concentrations of cytochalasin D actually induced fibrinogen binding to ␣IIb␤3 on unstimulated cells. Thus, our observations suggest that both PKC and the actin cytoskeleton play a role in regulating both the avidity and affinity of ␣IIb␤3 for ligands. How this might occur is uncertain. Platelet stimulation results in a conformational change in ␣IIb␤3 that increases its affinity for ligands (42,43). It is also associated with the disassembly of polymerized actin, followed by actin reassembly and a change in platelet morphology (44). It is conceivable that low concentrations of cytochalasin D can initiate actin disassembly, resulting in increases in both integrin mobility and affinity. Fox et al. (44) found that a variable amount of the ␣IIb␤3 in detergent lysates of unstimulated platelets was recovered with fragments of the membrane skeleton and was redistributed to a detergent-insoluble fraction containing a network of cytoplasmic actin filaments after ligand binding. They also observed that high concentrations of cytochalasin E inhibited the binding of the activation-dependent mAb PAC1 to ␣IIb␤3 on ADP and thrombin-stimulated platelets (26). Thus, it is possible that the membrane skeleton in platelets, or in our case in lymphocytes, interacts with the cytoplasmic tails of ␣IIb␤3 to constrain the integrin in a low affinity configuration. Relief of this constraint by agonists (or cytochalasins) could then be responsible for an augmented interaction of ␣IIb␤3 with immobilized fibrinogen and for its ability to bind soluble ligands.
Platelet stimulation is also associated with the formation of clusters of ligand-occupied ␣IIb␤3 on the platelet surface (26). However, ligand valency does not appear to be a factor in the ability of ␣IIb␤3 to recognize soluble ligands (45). Moreover, ␣IIb␤3 is a univalent receptor (11) and electron microscopy of fibrinogen bound to ␣IIb␤3 suggests that a fibrinogen molecule can only bind to one ␣IIb␤3 heterodimer on the surface of a given platelet (46). Hence, it is unlikely that clustering of ␣IIb␤3 alone can account for its ability to bind soluble fibrinogen. Nevertheless, it is possible, and even likely, that agonistinduced clustering of ␣IIb␤3 contributes to the augmented adherence of stimulated lymphocytes and platelets to immobilized fibrinogen.
Activity of members of the Ras family of small GTPases has major effects on cytoskeletal organization. For example, microinjection of activated forms of the small GTPases cdc42, Rac, and Rho into Swiss 3T3 fibroblasts results in the formation of filopodia, lamellopodia, and stress fibers, respectively (27). Similar effects have been reported in macrophages (47). Moreover, the small GTPase RhoA appears to play an important role in regulating integrin function in leukocytes, and perhaps in platelets as well. For example, inhibiting RhoA with C3 exoenzyme prevented PMA-stimulated ␣L␤2-mediated homotypic lymphocyte aggregation (14) and the fMLP-stimulated ␣4␤1mediated adherence of murine lymphocytes to VCAM-1 (16). Morii and co-workers (28) have reported that C3 exoenzyme completely inhibits thrombin and PMA-stimulated human platelet aggregation, although they found little correlation between the extent of RhoA inhibition by the C3 exoenzyme and the extent to which platelet function was impaired. On the other hand, in preliminary studies, Leng et al. (48) did not observe the reported effect of C3 exoenzyme on platelet aggregation. We found that C3 exoenzyme had no effect on either PMA and fMLP-stimulated lymphocyte adherence to fibrinogen, despite substantial ADP-ribosylation of RhoA. Thus, RhoA is unlikely to transduce the signals that regulate ␣IIb␤3 function, at least in our system. A more likely candidate is Rac1. Microinjection of activated Rac1 into Swiss 3T3 cells induces the submembranous accumulation of actin and the formation of integrin-containing focal complexes at the cell margin (49). Furthermore, activation of Rac1 by the exchange factor Tiam1 in T-lymphoma cells induces the formation of submembranous actin filaments, membrane ruffling, and an invasive phenotype (50). Thus, it is conceivable that a Rac-mediated reorganization of the membrane skeleton in lymphocytes, and by extrapolation in platelets, could be an intermediary step in the regulation of ␣IIb␤3 function by agonists.
In summary, we have shown that the ability of integrin ␣IIb␤3, expressed in B lymphocytes, to interact with immobilized and soluble fibrinogen can be stimulated by the G proteincoupled formyl peptide receptor. Moreover, our studies begin to define a signaling pathway that includes a PTX-inhibitable G-protein, PKC, and the actin cytoskeleton.


Segregation analysis of epithelial ovarian cancer in Finland.

Epithelial ovarian cancer is known to aggregate in families. The dominantly inherited ovarian cancer predisposing genes, BRCA1, BRCA2 and genes involved in the hereditary non-polyposis colorectal cancer (HNPCC) syndrome, have recently been identified. However, in the majority of families with more than one case of ovarian cancer, dominant inheritance cannot be recognized. We investigated familial clustering of epithelial ovarian cancer in a population-based sample of 663 Finnish ovarian cancer patients. A segregation analysis with the POINTER software was conducted on the 937 nuclear families from these 663 pedigrees. The major gene model was favoured, and the sporadic and multifactorial models were strongly rejected. In the studied population, the best fitting model was a recessive mode of inheritance, and 8% of ovarian cancer patients were estimated to be homozygous for the deleterious genotype. This evidence for recessively inherited ovarian cancer predisposition should be interpreted cautiously, as the analysis is subject to certain errors, which are discussed in the article. Results of this analysis, however, strongly emphasize the role of genetic factors in all familial aggregation of epithelial ovarian cancer.

Epithelial ovarian cancer aggregates in families. Evidence for this comes from two sources: epidemiological case-control studies, in which the first-degree relatives of ovarian cancer patients have been observed to have a twoto fivefold increased risk for ovarian cancer (Hartge et al, 1989;Schildkraut et al, 1989;Parazzini et al, 1992;Houlston et al, 1993;Goldgar et al, 1994;Hartge et al, 1994;Kerber and Slattery, 1995); and from family studies, which have identified families prone to ovarian cancer or, more commonly, to ovarian and breast cancer (Liber, 1950;Lewis and Clare Davidson, 1969;Li et al, 1970;Lynch et al, 1974;Fraumeni et al, 1975;Thor et al, 1976).
In family studies, cancer predisposition appears to be transmitted as an autosomal dominant trait, and a segregation analysis on ovarian cancer, based on data obtained from healthy women attending a family cancer unit, supports this observation (Houlston et al, 1991). The search for a dominantly inherited gene causing both breast and ovarian cancer predisposition has led to the isolation of the genes BRCAJ (Miki et al, 1994) and BRCA2 (Wooster et al, 1995), mutations of which have now been estimated to be involved in the majority of families with either site-specific ovarian cancer or breast-ovarian cancer predisposition (Steichen-Gersdorf et al, 1994;Narod et al, 1995).
Although families with multiple affected members stand out as examples of inherited cancer predisposition, the most common form of familial ovarian cancer is the occurrence of only two ovarian cancer cases in the family, without recognizible features of dominant inheritance (Greggi et al, 1990;Grover et al, 1993;Piver et al, 1993). This type of familial ovarian cancer contributes to the majority of the risk increase observed in the first-degree relatives Received 26 June 1997Revised 29 October 1997Accepted 30 October 1997 Correspondence to: Annika Auranen of ovarian cancer patients (Schildkraut and Thompson, 1988;Parazzini et al, 1992). To what extent familial ovarian cancer can be explained by mutations in the BRCAJ and BRCA2 genes or mutations in the other known dominantly inherited cancer-predisposing genes, most importantly those involved in the hereditary non-polyposis colorectal cancer (HNPCC; Aaltonen et al, 1994), is unresolved.
We have examined the familial occurrence of ovarian cancer among a Finnish population by conducting a complex segregation analysis (Lalouel and Morton, 1981) on an unselected sample of Finnish ovarian cancer patients. We have had the advantage of using data obtained from population registries and the Finnish Cancer Registry; therefore, the data used is completely verified from genealogical registries and medical records. With this population-based design, we were able to avoid major biases in relation to patient selection and cancer reporting.

MATERIALS AND METHODS
Ascertainment of the families The data on patients, from here on referred to as the probands, were obtained from the Finnish Cancer Registry. The populationbased and nationwide Finnish Cancer Registry has been operating since 1953 registering over 99% of all solid tumours in Finland (Teppo et al, 1994). All patients, who had an epithelial ovarian cancer diagnosed during the years 1980 to 1982 and who were under 76 years of age at the time of diagnosis were selected as probands. Patients with a borderline ovarian tumour were excluded. Altogether, there were 863 probands.
The method of data collection has been described in detail elsewhere (Auranen et al, 1996). Briefly, the local registries of the communities where the probands were born were contacted to obtain the names and birth dates of parents and siblings. In cases in which the family had moved to another community, tracing was continued until either of the parents deceased or the mother reached 50 years of age and further pregnancies were considered unlikely. Altogether, 700 (8 1%) of the probands' family members were traced. Failure to trace the family members resulted from inability to find the proband from the population registries of her reported birthplace (90 patients), lack of response from local officials (25 patients), born abroad (five patients) or failure to follow the family until additional children were considered unlikely, caused by the fact that the family changed location repeatedly (43 patients). In addition, data on the probands' husband and children were obtained from the parishes. The relatives of the 700 probands were followed up until death or to the end of 1993, whichever was first. The cancer morbidity of the relatives was checked from the files of the Finnish Cancer Registry. As the Finnish Cancer Registry has been operating only from the beginning of 1953, the cancer status of the male relatives deceased before this was classified as unknown. For mothers and sisters deceased during 1936-1952, death certificates were obtained, and their cancer classification was based on death certificate information. For women deceased before 1936, death certificates were not available, and their cancer status was classified as unknown. Cancer status was also classified as unknown for the 273 relatives, who were lost during the follow-up.
For 37 of the 700 probands, family information was incomplete in the sense that all female relatives were classified as having an unknown cancer status. As these families could not contribute to the analysis, they were excluded. In consequence, the final data set included 663 probands, who had the following relatives (number of unknown ovarian cancer phenotypes in brackets): 663 mothers (101), 663 fathers, 1339 sisters (153), 1333 brothers, 459 daughters (18) and 322 sons. The age of the daughters included in the analysis ranged from 20 to 68 years.

Segregation analysis
The 663 ovarian cancer pedigrees were partitioned into 937 nuclear families. The families were of two types: (1) sibships with the proband as a child (single selection, 663 families); and (2) children of the proband (complete selection, 274 families). Females below the age of 20 years were excluded from the genetic analysis. For single selection, the ascertainment probability pi was taken as 0.001. There were no pointers in the material.
Segregation analysis was carried out under a mixed model using the computer program POINTER (Lalouel and Morton, 1981). The mixed model assumes an underlying scale of genetic liability to which a major locus, polygenes and environmental factors operate. The effects are assumed to be independent and normally distributed. The parameters to be estimated are H: multifactorial heritability, q: gene frequency at the major locus; d: degree of dominance; and t: the displacement between the two heterozygous means. A recessive gene corresponds to d = 0 and a dominant to d = 1. Additivity corresponds to d = 0.5. The gene frequency was assumed to remain constant throughout all age classes.
Being affected was defined by a threshold on the liability scale. As liability to ovarian cancer varies with age, females were assigned to one of eight liability classes based on age (Table 1). All male relatives were given a morbid risk of 0.00001. The risk of ovarian cancer attributed to the jth liability class was defined by Iselius et al (1991): where I. is the cumulative incidence to the mid-point of j and M. is the cumulative specific mortality to the end of the preceding class. R. is therefore the probability that an individual observed in the jth class (dead or alive) is affected. The incidence (1) and mortality (M) of ovarian cancer were taken from the Finnish Cancer Registry for 1973-1982. Individuals with borderline ovarian tumours were considered normal in the present analysis.
For estimation of the parameters, conditional likelihood was used. To test between the hypotheses, minus twice the log likelihood (-21n L + C, where C is a constant) calculated under the general model was subtracted from the likelihood for a restricted model (with one or more parameters held constant). The difference is distributed as a chi-square with the appropriate number of degrees of freedom.
The penetrances (P) for different age groups were calculated using the formulas given by Iselius et  In the formula, G' stands for the disease gene.

RESULTS
Altogether, the 663 ovarian cancer probands had 31 first-degree relatives affected with ovarian cancer: 23 ovarian cancer probands had one affected relative and four probands had two affected relatives. In 23 of these 27 families, only sisters were affected. These families included three of the families with three ovarian cancers but, in one of these families, the mother had an abdominal cancer of unknown origin. In the analysis, she was defined as unaffected.
Breast cancer was present in 10 of the 23 families with two ovarian cancer cases but, in only four families, the age of the patient at the time of breast cancer diagnosis was less than 55 years. Breast cancer was not present in the four families with three ovarian cancer cases.
Of the 23 families with affected sisters only, in two families the mother had an unknown cancer phenotype and in one family the mother had breast cancer diagnosed at the age of 75 years. The remaining 20 mothers were not diagnosed with ovarian or breast cancer; all but one lived to be over 50 years of age and 13 of them over 70 years of age. Table 2 shows the results of the complex segregation analysis. The sporadic model, i.e. a model in which any familial occurrence of ovarian cancer is purely due to chance, was strongly rejected (x23= 41.55, P < 0.001). Also, the multifactorial model was rejected in favour of a major gene (x23= 14.12, P <0.001). The best fitting model was a recessive gene with a gene frequency of 0.0221. For all major gene models, H went to zero when iterated. There was no evidence for an additional familial component. We were not able to find any heterogeneity with regard to degree of dominance when the families were partitioned according to the mode of ascertainment and mating type (results not shown).
Characteristics of the major locus are given in Table 3. Lifetime penetrance for the deleterious genotype is estimated to be 90%. Taken over all liability classes, the probability of being homozygous for the recessive gene, given affection, is 0.079. The probability of an affected woman between 20 and 29 years of age having the deleterious recessive genotype is 0.57, while the corresponding probability is 0.06 for a woman in her seventies.

DISCUSSION
After the identification of the breast and ovarian cancer-predisposing genes, BRCAJ and BRCA2, it has been possible to demonstrate that a large majority of the families with both breast and ovarian cancer patients segregate mutations of these genes, especially BRCAI mutations (Narod et al, 1995). However, there is evidence from studies estimating the contribution of BRCAJ and BRCA2 genes to ovarian cancer incidence, that additional ovarian cancer-predisposing genes might exist Whittemore et al, 1997).
The results of this segregation analysis differ from the previously presented segregation analysis of ovarian cancer (Houlston et al, 1991) and from what is generally known of the inheritance of ovarian cancer (Claus and Schwartz, 1995) in the respect that a  recessive mode of inheritance is strongly favoured. The main result of this analysis is complementary to our previous analysis of this material using a different method (Auranen et al, 1996). We estimated the cancer incidence in these first-degree relatives of ovarian cancer patients and found that the incidence of ovarian cancer increased in sisters only. The incidence decreased both by the age of the sister and by the age of the proband at diagnosis, suggesting the involvement of a genetic component.
There are three main problems in this analysis that might have influenced the results. The first concerns possible underascertainment of ovarian cancer cases in the maternal generation. The cancer phenotype was unknown for 16% of the mothers; this, however, did not have a significant impact on the results. Additionally, 20% of the mothers had died before 1952. National cancer mortality and incidence statistics are available in Finland only from the beginning of 1953, which means that we were unable to estimate possible underdiagnosis of cancer in the maternal cohort. If there was severe underdiagnosis of ovarian cancer cases in these mothers, this has inevitably affected the results in favour of a recessive mode of inheritance.
The second problem relates to liability class estimates. We constructed liability classes accounting for morbid risks. A similar approach has also been used by a previous segregation analysis on ovarian cancer (Houlston et al, 1991). It would be advisable to construct liability classes on the basis of survival functions, but currently this is not possible with the POINTER software.
We have also used the same liability class estimates for both mother and proband generations, which means that we have not allowed for secular trends in ovarian cancer risk. The age-adjusted incidence rate of ovarian cancer in Finland has only slightly increased with time: it was 9.9 per 100 000 person-years in the 5year period 1961-1966 and 12.7 per 100 000 person-years in the 5-year period 1986-1990(Finnish Cancer Registry, 1995. As the age range of the probands in this study ranged from 20 to 75 years, the oldest probands were born before the mothers of the youngest probands. Instead of giving different baseline liability estimates for mother and proband generations, it would have been more justified to give liability estimates according to birth year. This was not considered to be necessary because of only slight changes in the ovarian cancer incidence rates over time. The third problem in our analysis is inability to take into account the occurrence of breast cancer in the family. When we estimated breast cancer incidence in this cohort (Auranen et al, 1996), the observed number of breast cancers was exactly the number that was expected, but there was some co-aggregation of breast and ovarian cancer. It is presumed that some of the families with two or more ovarian cancer cases harbour BRCAI or BRCA2 gene mutations, especially some of the ten families that also had a breast cancer case in a close relative. Because of the above problems, the results pointing to a recessive ovarian cancer predisposition should be interpreted cautiously. The possibility of recessive inheritance of ovarian cancer has only rarely been suggested previously, based either on consanguinity (Cramer et al, 1983) or on higher ovarian cancer mortality in sisters compared with mothers of ovarian cancer patients (Easton et al, 1996).
A detailed inspection of some of the previously published family studies reveals that families with more than one case of ovarian cancer can be roughly categorized into three groups: (1) families with ovarian and breast cancer in two or more generations (Li et al, 1970;Thor et al, 1976;Franceschi et al, 1982;Greggi et al, 1990;Narod et al, 1994); (2) families with only ovarian cancer in two or more generations (Liber, 1950;Lewis and Clare Davidson, 1969;Li et al, 1970;Fraumeni et al, 1975;Thor et al, 1976;Lurain and Piver, 1979); and (3) families with ovarian cancer in sisters only, without breast or other cancers (Kimbrough, 1929;Molloy, 1970;McCrann et al, 1974;Fraumeni et al, 1975;Skinner et al, 1977;Franceschi et al, 1982;Greggi et al, 1990;Narod et al, 1994). In the Gilda Radner Familial Ovarian Cancer Registry, mother-daughter relationships represented 50% of the families, but sister-sister relationship was the second most frequent type of relationship and represented 39% of all families (Piver et al, 1993).
It is possible that inherited predisposition to epithelial ovarian cancer is caused by heterogeneous mechanisms: the dominantly inherited cancer-predisposing genes, BRCAJ and BRCA2, which are most likely involved in families with breast cancer and families with ovarian cancer in two or more generations, and some other, possibly recessively inherited cancer-predisposing genes, in families without features of dominant inheritance. In support of this are the results from a segregation analysis of the CASH study, in which the existence of both shared and unique genes predisposing to breast and ovarian cancer were deduced (Schildkraut et al, 1989).
Another possibility is that mutations in the known ovarian cancer-predisposing genes, BRCAJ and BRCA2, have diversified effects on the phenotype of a mutation carrier, and the typical cancer families represent only the most severe forms of cancer susceptibility. If so, identification of BRCAJ and BRCA2 mutation carriers on the basis of family history of cancer, as well as genetic counselling of families, might be difficult.
The Finns are a small population that have lived as a genetic isolate for over 2000 years. As a result of this isolation, the genetic drift has led to the enrichment of certain recessive genes, whereas some genetic diseases common elsewhere have nearly disappeared (de la Chapelle, 1993). The results of this segregation analysis are, therefore, valid only in the Finnish population, and similar studies from other populations are needed. It would be especially important to analyse data that are not biased towards younger ovarian cancer probands, as has been the case in many of the previous studies (Schildkraut et al, 1989;Easton et al, 1996).
A search for BRCAI and BRCA2 gene mutations in the familial ovarian tumours in this material is currently under way. This should show us how many of these 27 families with multiple cases of ovarian cancer are affected by mutations in these genes. If BRCAJ or BRCA2 mutations are not detected in a considerable proportion of these familial ovarian tumours, molecular genetic studies aiming to find yet unidentified, possibly recessively inherited genes for ovarian cancer predisposition are greatly to be encouraged.