Channa Keshava

Distribution of HER2V655 genotypes in breast cancer cases and controls in the United States

The minor variant frequency of a HER2 polymorphism (HER2(V655)) has been determined for 471 United States women enrolled in a multiracial case-control study. Allelic frequencies varied significantly by race. Genotypic distributions showed no excess breast cancer risk associated with inheritance of HER2(V655) either as carriers (OR=1.2, 95% CI=0.8-1.9), heterozygotes (OR=1.2, 95% CI=0.8-1.9), or homozygotes (OR=1.4, 95% CI=0.4-4.2). Nor was there a significant association when each racial group was considered separately. The current study suggests the HER2(V655) allele is not a breast cancer risk factor for Caucasians, African-Americans, or Latinas.

Altered Gene Expression Patterns in MCF-7 Cells Induced by the Urban Dust Particulate Complex Mixture Standard Reference Material 1649a

Human exposures to polycyclic aromatic hydrocarbon (PAH) occur in complex mixtures. Here, gene expression patterns were investigated using standard reference material (SRM) 1649a (urban dust). MCF-7 cells were exposed to SRM 1649a alone or SRM 1649a with either benzo[a]pyrene (BP) or dibenzo[a,l]pyrene (DBP) for 24 hours. Global analyses of the gene expression data revealed alterations of 41 RNA transcripts with at least 2-fold change (signal log ratio </= -1 or >/= 1) in response to SRM 1649a exposure. Increase in expression of cytochrome P450 (CYP) genes was observed in response to BP exposure (CYP1A1 and CYP1B1; signal log ratio of 4.7 and 2.5, respectively). An additive induction of CYP1A1 and CYP1B1 was observed with cotreatment of SRM 1649a and BP. On the contrary, no change in gene expression of CYP1A1 and CYP1B1 was observed when the cells were exposed to DBP. Furthermore, to study the effect of complex PAH mixtures on the metabolic activation of carcinogenic PAH to DNA-binding derivatives and to relate this with gene expression studies, PAH-DNA adduct formation was determined. SRM 1649a decreased the total level of BP-DNA adducts in comparison with BP alone. No significant difference in adduct levels was observed in response to either DBP alone or in combination with SRM 1649a. These results provide a transcriptional signature for chemical carcinogen exposure; in addition, they suggest a major factor in carcinogenic activity of PAH within complex mixtures is their ability to promote or inhibit the activation of carcinogenic PAH by the induction of CYP enzymes.

DNA sequence variants of p53: cancer and aging.

To the Editor: p53 has a critical role in cell-cycle control. As such, it has been identified as an important target in human carcinogenesis. However, since human p53 was cloned, ⩾10 DNA-sequence polymorphisms have been identified (Matlashewski et al. 1987; Weston and Godbold 1997). The codon 72 polymorphism (arginine/proline: G/C), the first to be described, has been the subject of ⩾31 epidemiological case-control studies that have explored a potential association with cancer (Olschwang et al. 1991; Weston et al. 1992, 1994, 1997; Zhang et al. 1992; Kawajiri et al. 1993; Birgander et al. 1995, 1996b; Jin et al. 1995; Sjalander et al. 1995a, 1996a; Wu et al. 1995; Murata et al. 1996; To-Figueras et al. 1996; Golovleva et al. 1997; Weston and Godbold 1997; Yung et al. 1997; Hayes et al. 1998; Helland et al. 1998; Hildesheim et al. 1998; Josefsson et al. 1998; Lanham et al. 1998; Minaguchi et al. 1998; Rosenthal et al. 1998; Storey et al. 1998; Tagawa et al. 1998; Wang-Gohrke et al. 1998). Although they err on the side of caution by citing Weston and Godbold (1997), Bonafe et al. (1999, p. 293), referring to the codon 72 polymorphism, state that “overall, the available data in the literature suggest that p53 variants may be considered as risk factors for some of the major neoplastic diseases in humans, such as lung, colorectal, breast and cervical cancer and are expected to affect survival.” With respect to codon 72, we contend that this is probably not the case. In the available data, lung cancer is the subject of eight studies (Weston et al. 1992, 1994; Kawajiri et al. 1993; Birgander et al. 1995; Jin et al. 1995; Murata et al. 1996; To-Figueras et al. 1996; Tagawa et al. 1998). Three studies claim a statistically significant association: in one study, a subset analysis suggested a relationship in lung cancer cases diagnosed at age <53 years (Jin et al. 1995); in the other two, the allelic frequencies were almost identical (and the difference was not significant) between cases and controls (Kawajiri et al. [1993] observed a proline-allele frequency of .35 for controls and .36 for cases [n = 347 and 328, respectively]). Murata et al. (1996) observed a proline-allele frequency of .40 for controls and .35 for cases (n = 152 and 191, respectively), but associations of cancer risk were claimed on the basis of higher numbers of homozygotes (Kawajiri et al. 1993; Murata et al. 1996). One study (Kawajiri et al. 1993) implicated proline; the other (Murata et al. 1996), arginine. Most recently, the relationship between cervical cancer, human papillomavirus (HPV) infection, and inheritance of the arginine allele has received considerable attention. The initial study, cited by Bonafe et al. (1999), showed a high degree of correlation between inheritance of the arginine allele and cervical cancer risk when HPV infection was present but not when it was absent (Storey et al. 1998). There are now eight studies of cervical cancer, but only one shows any association and seven are null (Hayes et al. 1998; Helland et al. 1998; Hildesheim et al. 1998; Josefsson et al. 1998; Lanham et al. 1998; Minaguchi et al. 1998; Rosenthal et al. 1998). There are five breast cancer studies (Kawajiri et al. 1993; Sjalander et al. 1996a; Weston et al. 1997; Helland et al. 1998; Wang-Gohrke et al. 1998); only one reached significance. Of the other published studies, a positive association has been claimed in 0/2 for nasopharyngeal cancer (Birgander et al. 1996b; Golovleva et al. 1997; Yung et al. 1997), 0/4 for colon cancer (Olschwang et al. 1991; Kawajiri et al. 1993; Jin et al. 1995; Sjalander et al. 1995a), 0/2 for bladder/urologic cancer (Kawajiri et al. 1993; Wu et al. 1995), 0/1 for acute myelogenous leukemia (Zhang et al. 1992), and 1/1 for stomach cancer (Kawajiri et al. 1993). Again, for stomach cancer, the allelic frequencies were not different for cases and controls, and an association of cancer risk with the arginine variant was claimed on the basis of higher numbers of arginine homozygotes in cases (Kawajiri et al. 1993; this report used the same control group noted above, and the proline-allele frequency in stomach cancer was .28 [n=140]). We contend, therefore, that the balance of the results of human molecular epidemiological association studies suggests that codon 72 allelism does not have an impact on human cancer risk. Specifically, at most, only 6 of 31 positive associations have been reported, and none have received consistent support in attempts to replicate them (Kawajiri et al. 1993; Jin et al. 1995; Murata et al. 1996; Sjalander et al. 1996a; Storey et al. 1998). Moreover, there is no obvious, plausible biological basis for an association of cancer risk with the codon 72 polymorphism. The impact of proline on the tertiary structure of a protein is disruption of α-helices. A proline residue resides at the monomeric position codon 71; therefore, at codon 72 there is no obvious consequence to proline. Results were reported for p53, codon 72, genotype frequencies in healthy Italian centenarians and younger controls (Bonafe et al. 1999). The expectation was of allelic winnowing in the case of a codon 72 variant associated with cancer susceptibility and subsequent survival. The frequency of proline-allele carriers was .539 in controls and .506 in centenarians, 3.3% lower (and the corresponding allelic frequency was 2.7% lower, but the difference was not significant). We conjecture that their comparison of centenarians (n=176) with younger controls (n=204) is too crude to detect the predicted allelic winnowing (Bonafe et al. 1999). First, the cause of death from cancer in the Italian population is ∼23% (WHO 1987; Lopez 1990). Second, even though we do not agree that there is good evidence for an association between inheritance at codon 72 and cancer susceptibility, because of the molecular epidemiological data reviewed above, let us assume that the proline variant carries a relative risk of 1.5–2.0 (Kawajiri et al. 1993). On the basis of this assumption we might expect a reduction of 3.0%–4.8% in proline-allele carriers as the population ages (table 1 shows a simulated 2×2 table for a standard population of 1,000 persons, given a relative risk of 1.5 and a cancer death rate of 23% [WHO 1987; Lopez 1990]). In the letter by Bonafe et al. (1999), a reduction of 3.3% in the frequency of proline-allele carriers among centenarians was reported. This reduction is consistent with their hypothesis, although the authors suggest that it is not (Bonafe et al. 1999). Moreover, our corresponding projected reduction in allelic frequency is 2.8% for a relative risk of 1.5; Bonafe et al. (1999) observed a 2.7% reduction in frequency. However, to have, at a significance level of .05, 80% power to detect a 3.0%–4.8% reduction in the prevalence of proline-allele carriers, a minimum of 2,002–3,178 people would be needed, given a relative risk of 2.0; and 5,176–8,182 would be needed, given a relative risk of 1.5 (results of power calculations are given in table 2). Table 1 Simulated Relative-Risk Calculation for a Standard Population of 1,000 Persons[Note] Table 2 Power Calculations Notwithstanding these conclusions and observations, we believe that p53 allelism is an important cancer-susceptibility factor but that it is more complex than that simply defined by the polymorphism at codon 72. A series of studies by a Swedish group (Beckman et al. 1994; Birgander et al. 1995, 1996a, 1996b; Sjalander et al. 1995a, 1995b, 1996a, 1996b) indicated that a constellation of three p53 polymorphisms (intron 3, codon 72, and intron 6) constituted a haplotype predictive of increased breast cancer risk (odds ratio [OR] = 2.9, 95% confidence interval [CI] = 1.4–6.3 [Sjalander et al. 1996a]). Haplotypes were deduced on the basis of population frequencies of the individual polymorphisms. First, estimates of pairwise haplotype frequencies were calculated, and extended haplotype frequencies were deduced from this information. A subsequent study examined the same question but used a PCR-based method for physical determination of the haplotypes (Weston et al. 1997, 1998). The results of the latter study were consistent with those reported in the study by Sjalander et al. (1996a) (OR = 2.5, 95% CI = 1.3–4.8, in postmenopausal women) and further implicated a specific haplotype, designated “p531-2-1.” More recently, a third study, estimating extended haplotypes of these three polymorphisms in a German population, provided results consistent with an elevated breast cancer risk associated with inheritance of p531-2-1 (OR = 2.0, 95% CI = 1.0–3.9 [Wang-Gohrke et al. 1998]). It is to be noted that this latest report has adopted an allelic nomenclature different from that developed by Beckman et al. (1994) and adopted by others (Sjalander et al. 1996a; Weston et al. 1997, 1998). The balance of the results of human breast cancer studies of molecular epidemiological haplotype association—specifically, three positive associations in three studies—suggests that inheritance of the p531-2-1 haplotype does have an impact on human breast cancer risk (Sjalander et al. 1996a; Wang-Gohrke et al. 1998; Weston et al. 1998). However, more research is needed to resolve this question. In our laboratory, we have adopted a complementary molecular epidemiological and basic-science approach. First, we are trying again to replicate the epidemiological studies of breast cancer, by using the PCR-based physical haplotype method in a population-based case-control study that has sufficient power. Second, we have derived normal human mammary cell strains with known haplotypes and plan to test their response to suspect breast carcinogens.

Genomic instability in silica- and cadmium chloride-transformed BALB/c-3T3 and tumor cell lines by random amplified polymorphic DNA analysis

Our earlier studies using random amplified polymorphic DNA (RAPD) analysis have shown genetic instability in human lung cancer tissues. Here we have investigated the potential for genetic instability in silica- and cadmium chloride (CdCl2)-transformed BALB/c-3T3 cell lines. Non-transformed, transformed BALB/c-3T3 cells, and tumor cell lines (obtained by injecting nude mice with transformed cell lines) were analyzed for genomic changes. DNAs from 10 different transformed clones and their corresponding tumor cell lines were amplified individually by RAPD analysis using 10 arbitrary primers. DNA from non-transformed BALB/c-3T3 cells was used as a control to compare genetic alterations, if any, between non-transformed, transformed and tumor cell populations. PCR products from RAPD were electrophoretically separated on agarose gels and the banding profiles were visualized by ethidium bromide staining. Five of the 10 primers tested revealed genomic changes in silica-transformed cell lines when compared to non-transformed BALB/c-3T3 cells. Comparison of all 10 transformed and tumor cell lines showed varied degrees of genomic changes using all 10 primers. CdCl2-transformed cell lines displayed fewer genomic changes, only three of 10 primers showed a positive result. CdCl2-transformed cells and their corresponding tumor cell lines showed specific banding pattern differences in six of the 10 samples tested with six of the 10 primers. Changes in band intensity were the most commonly observed changes both in silica- and CdCl2-transformed and tumor cell lines. The results seem to indicate a progressive change in genomic rearrangements which may directly or indirectly be associated with progression of tumorigenesis.

CYP1A1 and CYP1B1 gene expression and DNA adduct formation in normal human mammary epithelial cells exposed to benzo[a]pyrene in the absence or presence of chlorophyllin

Benzo[a]pyrene (BP) is a potent pro-carcinogen and ubiquitous environmental pollutant. Here, we examined the induction and modulation of CYP1A1 and CYP1B1 and 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct formation in DNA from 20 primary normal human mammary epithelial cell (NHMEC) strains exposed to BP (4muM) in the absence or presence of chlorophyllin (5muM). Real-time polymerase chain reaction (RT-PCR) analysis revealed strong induction of both CYP1A1 and CYP1B1 by BP, with high levels of inter-individual variability. Variable BPdG formation was found in all strains by r7, t8-dihydroxy-t-9, 10 epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence assay (CIA). Chlorophyllin mitigated BP-induced CYP1A1 and CYP1B1 gene expression in all 20 strains when administered with BP. Chlorophyllin, administered prior to BP-exposure, mitigated CYP1A1 expression in 18/20 NHMEC strains (p<0.005) and CYP1B1 expression in 17/20 NHMEC strains (p<0.005). Maximum percent reductions of CYP1A1 and CYP1B1 gene expression and BPdG adduct formation were observed when cells were pre-dosed with chlorophyllin followed by administration of the carcinogen with chlorophyllin (p<0.005 for CYP1A1 and CYP1B1 expression and p<0.0005 for BPdG adducts). Therefore, chlorophyllin is likely to be a good chemoprotective agent for a large proportion of the human population.

Transcriptional profiles of benzo(a)pyrene exposure in normal human mammary epithelial cells in the absence or presence of chlorophyllin

Benzo(a)pyrene (BP) exposure causes alterations in gene expression in normal human mammary epithelial cells (NHMECs). This study used Affymetrix Hu-Gene133A arrays, with 14,500 genes represented, to evaluate modulation of BP-induced gene expression by chlorophyllin in six NHMEC strains derived from different donors. A major goal was to seek potential biomarkers of carcinogen exposure and how they behave in the presence of a chemopreventive agent. NHMECs (passage 6 and 70% confluence) were exposed for 24h to either vehicle control, or BP, or chlorophyllin followed by BP and chlorophyllin together. BP exposure resulted in approximately 3-fold altered expression of 49 genes in at least one of the six NHMEC strains. When cells were exposed to chlorophyllin pre-treatment followed by BP plus chlorophyllin, expression of 125 genes was similarly altered. Genes in the functional categories of xenobiotic metabolism, cell signaling, cell motility, cell proliferation, cellular transcription, metabolism, cell cycle control, apoptosis and DNA repair were identified. Only CYP1B1 and ALDH1A3 were consistently up-regulated by approximately 3-fold in most of the cell strains (at least 4) when exposed to BP. Cluster analysis identified a suite of 13 genes induced by BP where induction was mitigated in the presence of chlorophyllin. Additionally, cluster analysis identified a suite of 16 genes down-regulated by BP where induction was partially restored in the presence of chlorophyllin.

Reduction in Tamoxifen-Induced CYP3A2 Expression and DNA Adducts Using Antisense Technology

Tamoxifen (TAM) is widely used in the treatment and prevention of breast cancer. There is clear evidence that cytochrome P450 (CYP) 3A enzymes play an important role in TAM metabolism, resulting in metabolites that lead to formation of TAM–DNA adducts. We have investigated the effect of CYP3A2 antisense (AVI‐4472) exposure on CYP3A2 transcription, enzyme activity, translation, and TAM–DNA adducts, in livers of rats administered TAM (50 mg/kg body weight [bw]/day) for 7 days. The study design included administration of 0, 0.5, 2.5, or 12.5 mg AVI‐4472/kg bw/day for 8 days, beginning 1 day before TAM exposure. The specific activity of CYP3A2 was increased after TAM administration, and decreased significantly (∼70%) in the presence of 12.5 mg AVI‐4472. CYP3A2 protein levels, determined by immunoblot analysis, showed a similar pattern. Hepatic TAM–DNA adduct levels were measurable in all TAM‐exposed groups. However, when rats were co‐treated with 2.5 and 12.5 mg AVI‐4472/kg bw/day, statistically significant (∼50%) reductions in TAM–DNA adduct levels (2.0–2.8 adducts/108 nucleotides) were observed compared to rats treated with TAM alone (5.1 adducts/108 nucleotides). Rat toxicology U34 arrays (Affymetrix) were used to investigate the modulation of gene expression patterns on co‐administration of TAM with AVI‐4472. Results indicated that several CYP genes were down regulated although no significant induction of CYP3A2 was observed in the TAM‐exposed rats co‐treated with AVI‐4472. Overall the data suggest the utility of antisense technology in the redirection of TAM metabolism thereby lowering TAM genotoxicity in rat liver. Published 2005 Wiley‐Liss, Inc.