Chromosomal instability is a feature of many different mendelian cancer susceptibility syndromes as well as acquired tumors (1) and may be induced by a variety of physical, chemical, and biological agents. Although chromosomal instability induced by ionizing radiation or ultraviolet radiation has been studied extensively, there is relatively little information on the molecular factors that protect cells against damage by other genotoxic agents. A number of such agents are known to induce apoptosis mediated by p53 (2) in collaboration with other cellular proteins (3); such protein-protein interactions determine the sensitivity of cells to genotoxic agents and antineoplastic drugs.Fanconi anemia (FA) 1 is a human disease model for chromosomal instability and hypersensitivity to genotoxic agents. Cells from FA patients have unstable chromosomes and are highly susceptible to apoptosis after exposure to specific clastogens, particularly bifunctional cross-linkers, such as mitomycin C (MMC) (4). The phenotype of FA is pleiotropic and includes birth defects, bone marrow failure, and cancer predisposition. Somatic cell fusion studies have identified eight distinct complementation groups, designated A-H, and the genes defective in FA complementation groups A (FANCA) (5, 6), C (FANCC) (7), and G (FANCG) (8) have been cloned. Although all three genes were cloned by virtue of their ability to suppress the toxicity of MMC in FA lymphoblastoid cells of appropriate complementation groups, the encoded proteins have no significant homology to each other or to other known proteins, save for limited homology between FANCA and heme peroxidases (9). Our previous data as well as data from other laboratories have shown that FANCC is predominantly cytoplasmic (10 -12), but others have reported that a fraction of FANCC is also nuclear (12, 13). FANCA is both nuclear and cytoplasmic (13-15). Forced targeting of these proteins to particular subcellular compartments has revealed that FANCC requires cytoplasmic localization (16) and FANCA nuclear localization (14) to be able to suppress cross-linker-induced cytotoxicity. Furthermore, the function of FANCC has been partially elucidated by its interaction with the microsomal membrane protein NADPH cytochrome P-450 reductase, an enzyme involved in the metabolic activation of many xenobiotics, including MMC (17). The function of other FA proteins, however, remains elusive and may involve a variety of functions including DNA repair, detoxification, and regulation of apoptosis (4). We (18) and others (19) have recently described an interaction between FANCA and FANCG within protein complexes found in non-FA but not in FA cells from several complementation groups. However, FANCA-FANCG complexes were not universally absent in all FA subgroups, and in the latter case the complexes were presumed to be functionally deficient. Therefore, a direct link between the expression of FANCA-FANCG complexes and cellular resistance to MMC could not be established. Also, except for an amino-terminal domain of FAN...
Genetic differences that underlie inter-individual variation in the metabolism of common carcinogens are important potential sources of cancer susceptibility. Cytochrome P450 1B1 (CYP1B1), a central enzyme in the activation of the ubiquitous environmental carcinogen benzo[a]pyrene (B[a]P), has several genetic variants. This study investigated six rare mutations and four common polymorphisms for their effects on B[a]P metabolism. Five missense mutations associated with congenital glaucoma (Gly61Glu, Gly365Trp, Asp374Asn, Pro437Leu and Arg469Tryp) dramatically decreased the capacity of CYP1B1 to convert (-)benzo[a]pyrene-7R-trans-7,8-dihyrodiol (B[a]P-7,8-diol) to (+/-)benzo[a]pyrene-r-7,t-8-dihydrodiol-9,10-epoxides. These five mutations resulted in enzymes with 3-12% of normal activity when assayed in vitro using an Saccharomyces cerevisiae microsomal expression system. A 10 bp deletion mutation produced no detectable protein or activity. In contrast, proteins containing all possible combinations of four common single nucleotide polymorphisms (Arg48Gly, Ala199Ser, Val432Leu, Asn453Ser) had modest effects on B[a]P-7,8-diol metabolism. Michaelis-Menten analysis suggested that two alleles, Arg48, Ala119, Val432, Ser453 (RAVS) and Arg48, Ala119, Leu432, Ser453 (RALS), have KM values 2-fold lower than Arg48, Ala119, Val432, Ser453 (RAVN): 1.4+/-0.3 and 1.3+/-0.4 microM, respectively, compared with 2.8+/-0.8 microM (P<0.05). However, these differences could not be confirmed with direct measurements of rate at low substrate concentration. There were no significant differences for either of two other kinetic parameters, kcat or kcat/KM. Allele frequency analysis in three populations reveals the Ser453 variant is rare among those of Asian (<1%) and African ancestry (<4%), and more common in individuals of European ancestry (16%). Haplotypes containing the Ser453 variant were uncommon; only RALS was detectable in our small populations. The RALS allele occurred between 0.5% in Asians and 15% in Europeans. Our study demonstrates that rare, disease-associated mutations in CYP1B1 significantly decrease the enzyme's metabolism of B[a]P-7,8-diol; however, our results do not identify any major differences in this metabolism due to four common single amino acid polymorphisms.
Seckel syndrome is a rare autosomal recessive disorder. The classical presentation includes pre- and postnatal growth deficiency, mental retardation, and characteristic facial appearance. There have been several reports of associated hematological abnormalities and chromosomal breakage, findings suggestive of Fanconi anemia (FA). We tested for these findings in two Arabic patients with this syndrome. We compared the growth profile of lymphoblastoid cells from our patients and their parents with the FA group A cell line HSC72 in the presence and absence of mitomycin C (MMC). By Western analysis, we also determined the expression of FAA and FAC, two FA disease gene products that together account for approximately 80% of FA. Unlike HSC72 cells, cells from the patients were resistant to MMC, and both FAA and FAC proteins were expressed at similar levels in all cell lines. There is an increasing recognition of clinical variability and perhaps genetic heterogeneity in Seckel syndrome. Our results demonstrate that cross-link sensitivity comparable to FA is not a uniform finding in patients with Seckel syndrome.
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