Cancer-Specific Alterations in Nuclear Matrix Proteins Determined by Multi-omics Analyses of Ductal Carcinoma in Situ.

Breast cancer (BC) is the most common cancer affecting women in the United States. Ductal carcinoma in situ (DCIS) is the earliest identifiable pre-invasive BC lesion. Estimates show that 14 to 50% of DCIS cases progress to invasive BC. Our objective was to identify nuclear matrix proteins (NMP) with specifically altered expression in DCIS and later stages of BC compared to non-diseased breast reduction mammoplasty and a contralateral breast explant using mass spectrometry and RNA sequencing to accurately identify aggressive DCIS. Sixty NMPs were significantly differentially expressed between the DCIS and non-diseased breast epithelium in an isogenic contralateral pair of patient-derived extended explants. Ten of the sixty showed significant mRNA expression level differences that matched the protein expression. These 10 proteins were similarly expressed in non-diseased breast reduction cells. Three NMPs (RPL7A, RPL11, RPL31) were significantly upregulated in DCIS and all other BC stages compared to the matching contralateral breast culture and an unrelated non-diseased breast reduction culture. RNA sequencing analyses showed that these three genes were upregulated increasingly with BC progression. Finally, we identified three NMPs (AHNAK, CDC37 and DNAJB1) that were significantly downregulated in DCIS and all other BC stages compared to the isogenically matched contralateral culture and the non-diseased breast reduction culture using both proteomics and RNA sequencing techniques.

Nucleotide excision repair deficiency is intrinsic in sporadic stage I breast cancer.

The molecular etiology of breast cancer has proven to be remarkably complex. Most individual oncogenes are disregulated in only approximately 30% of breast tumors, indicating that either very few molecular alterations are common to the majority of breast cancers, or that they have not yet been identified. In striking contrast, we now show that 19 of 19 stage I breast tumors tested with the functional unscheduled DNA synthesis assay exhibited a significant deficiency of DNA nucleotide excision repair (NER) capacity relative to normal epithelial tissue from disease-free controls (n = 23). Loss of DNA repair capacity, including the complex, damage-comprehensive NER pathway, results in genomic instability, a hallmark of carcinogenesis. By microarray analysis, mRNA expression levels for 20 canonical NER genes were reduced in representative tumor samples versus normal. Significant reductions were observed in 19 of these genes analyzed by the more sensitive method of RNase protection. These results were confirmed at the protein level for five NER gene products. Taken together, these data suggest that NER deficiency may play an important role in the etiology of sporadic breast cancer, and that early-stage breast cancer may be intrinsically susceptible to genotoxic chemotherapeutic agents, such as cis-platinum, whose damage is remediated by NER. In addition, reduced NER capacity, or reduced expression of NER genes, could provide a basis for the development of biomarkers for the identification of tumorigenic breast epithelium.

Identification of Hydroxysteroid (17ß) dehydrogenase type 12 (HSD17B12) as a CD8+ T-cell-defined human tumor antigen of human carcinomas.

Hydroxysteroid (17ß) dehydrogenase type 12 (HSD17B12) is a multifunctional isoenzyme functional in the conversion of estrone to estradiol (E2), and elongation of long-chain fatty acids, in particular the conversion of palmitic to archadonic (AA) acid, the precursor of sterols and the inflammatory mediator, prostaglandin E(2). Its overexpression together with that of COX-2 in breast carcinoma is associated with a poor prognosis. We have identified the HSD17B12(114-122) peptide (IYDKIKTGL) as a naturally presented HLA-A*0201 (HLA-A2)-restricted CD8(+) T-cell-defined epitope. The HSD17B12(114-122) peptide, however, is poorly immunogenic in its in vitro ability to induce peptide-specific CD8(+) T cells. Acting as an "optimized peptide", a peptide (TYDKIKTGL), which is identical to the HSD17B12(114-122) peptide except for threonine at residue 1, was required for inducing in vitro the expansion of CD8(+) T-cell effectors cross-reactive against the HSD17B12(114-122) peptide. In IFN-γ ELISPOT assays, these effector cells recognize HSD17B12(114-122) peptide-pulsed target cells, as well as HLA-A2(+) squamous cell carcinoma of the head and neck (SCCHN) and breast carcinoma cell lines overexpressing HSD17B12 and naturally presenting the epitope. Whereas growth inhibition of a breast carcinoma cell line induced by HSD17B12 knockdown was only reversed by AA, in a similar manner, the growth inhibition of the SCCHN PCI-13 cell line by HSD17B12 knockdown was reversed by E2 and AA. Our findings provide the basis for future studies aimed at developing cancer vaccines for targeting HSD17B12, which apparently can be functional in critical metabolic pathways involved in inflammation and cancer.

Permanently blocked stem cells derived from breast cancer cell lines.

Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. However, an unanticipated result was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immunocompromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs.

Elevated somatic mutation and evidence of genomic instability in veterans with Gulf War illness.

AIMS: Gulf War illness (GWI) is thought to be associated with exposures experienced by soldiers deployed in the 1991 Gulf War. A major question is how these exposures continue to influence the health of these individuals three decades later. One potentially permanent effect of such exposures is the induction of genetic mutations. We investigated whether veterans with GWI exhibited persistently elevated levels of somatic mutation. MATERIALS AND METHODS: We applied the blood-based glycophorin A (GPA) somatic mutation assay to a cohort of veterans diagnosed with GWI and a set of both concurrent and historic age-matched controls. This assay quantifies red blood cells with a phenotype consistent with loss of one allele at the genetic determinant for the MN blood group, the GPA gene. KEY FINDINGS: As a population, those affected with GWI exhibited an uninduced mutation frequency at the GPA locus that was effectively twice that observed in controls, a result that was statistically significant. This result was influenced by an increase in the incidence of individuals with aberrantly high mutation frequencies, seemingly higher than would be expected by dose extrapolation and consistent with the induction of localized genomic instability in the hematopoietic bone marrow stem cells. When these "outliers" were removed from consideration, the remaining affected population retained a significantly higher mean allele loss mutation frequency, suggesting that both dose-dependent bone marrow genotoxicity and induction of genomic instability are contributing to the elevation in mutation frequency in these affected veterans. SIGNIFICANCE: This study provides evidence that manifestation of GWI is associated with increased cumulative exposure to agents capable of inducing persistent mutations in bone marrow stem cells. Whether these mutations are involved in the clinical aspects of the condition or are simply biomarkers of overall exposure has yet to be determined. The increased incidence of genomic instability suggests that this persistent mutation can have important delayed effects on cellular integrity.

Molecular Analysis of Mutations in the Human HPRT Gene.

The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.

Preliminary Evidence for a Hormetic Effect on DNA Nucleotide Excision Repair in Veterans with Gulf War Illness.

INTRODUCTION: Veterans of the 1991 Gulf War were potentially exposed to a mixture of stress, chemicals and radiation that may have contributed to the persistent symptoms of Gulf War Illness (GWI). The genotoxic effects of some of these exposures are mediated by the DNA nucleotide excision repair (NER) pathway. We hypothesized that individuals with relatively low DNA repair capacity would suffer greater damage from cumulative genotoxic exposures, some of which would persist, causing ongoing problems. MATERIALS AND METHODS: Blood samples were obtained from symptomatic Gulf War veterans and age-matched controls. The unscheduled DNA synthesis assay, a functional measurement of NER capacity, was performed on cultured lymphocytes, and lymphocyte mRNA was extracted and analyzed by sequencing. RESULTS: Despite our hypothesis that GWI would be associated with DNA repair deficiency, NER capacity in lymphocytes from affected GWI veterans actually exhibited a significantly elevated level of DNA repair (p = 0.016). Both total gene expression and NER gene expression successfully differentiated individuals with GWI from unaffected controls. The observed functional increase in DNA repair capacity was accompanied by an overexpression of genes in the NER pathway, as determined by RNA sequencing analysis. CONCLUSION: We suggest that the observed elevations in DNA repair capacity and NER gene expression are indicative of a "hormetic," i.e., induced or adaptive protective response to battlefield exposures. Normally such effects are short-term, but in these individuals this response has resulted in a long-term metabolic shift that may also be responsible for the persistent symptoms of GWI.

Melatonin and breast cancer: cellular mechanisms, clinical studies and future perspectives.

Recent studies have suggested that the pineal hormone melatonin may protect against breast cancer, and the mechanisms underlying its actions are becoming clearer. Melatonin works through receptors and distinct second messenger pathways to reduce cellular proliferation and to induce cellular differentiation. In addition, independently of receptors melatonin can modulate oestrogen-dependent pathways and reduce free-radical formation, thus preventing mutation and cellular toxicity. The fact that melatonin works through a myriad of signalling cascades that are protective to cells makes this hormone a good candidate for use in the clinic for the prevention and/or treatment of cancer. This review summarises cellular mechanisms governing the action of melatonin and then considers the potential use of melatonin in breast cancer prevention and treatment, with an emphasis on improving clinical outcomes.

(Z)-1,1-Dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane induces concentration-dependent growth inhibition, apoptosis, and coordinates regulation of apoptotic genes in TRAMP cells.

(Z)-1-1-Dichloro-2,3-diphenylcyclopropane (A(II)) and (Z)-1,1-dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane [2-(4-methoxyphenyl)-A(II)] inhibit tubulin polymerization, PSA production, and the proliferation of human prostate cancer cells. The actions of the agents were studied in three transgenic adenocarcinomas of the mouse prostate (TRAMP) cell lines. Antiproliferative potencies were determined and cells treated with the more potent 2-(4-methoxyphenyl)-A(II) were examined for induction of apoptosis. Microarray analyses were conducted to determine the apoptosis-related genes up- and down-regulated by the agent. 2-(4-Methoxyphenyl)-A(II) concentration-dependently inhibited growth of all three cell lines. Fifty percent and 100% growth inhibitory and 50% lethal concentrations were determined to be 0.3, 1.5, and 5 muM, respectively. Minimum detectable apoptosis-inducing concentrations by ELISA were 0.10 to 0.14 muM. PARP cleavage and two-color flow cytometry assays verified apoptosis induction. Microarray analyses showed Bok and Siva-pending to be up-regulated and that Birc, Dad1, and Atf5 were down-regulated. 2-(4-methoxyphenyl)-A(II) inhibits proliferation and induces apoptosis in the in vivo-adaptable TRAMP cells, suggesting the compound should be further examined in preclinical models.

Nucleotide excision repair is a predictor of early relapse in pediatric acute lymphoblastic leukemia.

BACKGROUND: Nucleotide Excision Repair (NER) is a major pathway of mammalian DNA repair that is associated with drug resistance and has not been well characterized in acute lymphoblastic leukemia (ALL). The objective of this study was to explore the role of NER in relapsed ALL patients. We hypothesized that increased expression of NER genes was associated with drug resistance and relapse in ALL. METHODS: We performed secondary data analysis on two sets of pediatric ALL patients that all ultimately relapsed, and who had matched diagnosis-relapse gene expression microarray data (GSE28460 and GSE18497). GSE28460 included 49 precursor-B-ALL patients, and GSE18497 included 27 precursor-B-ALL and 14 T-ALL patients. Microarray data were processed using the Plier 16 algorithm and the 20 canonical NER genes were extracted. Comparisons were made between time of diagnosis and relapse, and between early and late relapsing subgroups. The Chi-square test was used to evaluate whether NER gene expression was altered at the level of the entire pathway and individual gene expression was compared using t-tests. RESULTS: We found that gene expression of the NER pathway was significantly increased upon relapse in patients that took 3 years or greater to relapse (late relapsers, P = .007), whereas no such change was evident in patients that relapsed in less than 3 years (early relapsers, P = .180). Moreover, at diagnosis, the NER gene expression of the early relapsing subpopulation was already significantly elevated over that of the late relapsing group (P < .001). This pattern was validated by an 'NER score' established by averaging the relative expression of the 20 canonical NER genes. The NER score at diagnosis was found to be significantly associated with disease-free survival in precursor-B-ALL (P < .001). CONCLUSION: Patients are over two times more likely to undergo early relapse if they have a high NER score at diagnosis, hazard ratio 2.008, 95% CI (1.256-3.211). The NER score may provide a underlying mechanism for "time to remission", a known prognostic factor in ALL, and a rationale for differential treatment.

Elevated levels of somatic mutation in a manifesting BRCA1 mutation carrier.

Homozygous loss of activity at the breast cancerpredisposing genes BRCA1 and BRCA2 (FANCD1) confers increased susceptibility to DNA double strand breaks, but this genotype occurs only in the tumor itself, following loss of heterozygosity at one of these loci. Thus, if these genes play a role in tumor etiology as opposed to tumor progression, they must manifest a heterozygous phenotype at the cellular level. To investigate the potential consequences of somatic heterozygosity for a BRCA1 mutation demonstrably associated with breast carcinogenesis on background somatic mutational burden, we applied the two standard assays of in vivo human somatic mutation to blood samples from a manifesting carrier of the Q1200X mutation in BRCA1 whose tumor was uniquely ascertained through an MRI screening study. The patient had an allele-loss mutation frequency of 19.4 x 10(-6) at the autosomal GPA locus in erythrocytes and 17.1 x 10(-6) at the X-linked HPRT locus in lymphocytes. Both of these mutation frequencies are significantly higher than expected from age-matched disease-free controls (P < 0.05). Mutation at the HPRT locus was similarly elevated in lymphoblastoid cell lines established from three other BRCA1 mutation carriers with breast cancer. Our patient's GPA mutation frequency is below the level established for diagnosis of homozygous Fanconi anemia patients, but consistent with data from obligate heterozygotes. The increased HPRT mutation frequency is more reminiscent of data from patients with xeroderma pigmentosum, a disease characterized by UV sensitivity and deficiency in the nucleotide excision pathway of DNA repair. Therefore, this BRCA1-associated breast cancer patient manifests a unique phenotype of increased background mutagenesis that likely contributed to the development of her disease independent of loss of heterozygosity at the susceptibility locus.

Prospective screening study of 0.5 Tesla dedicated magnetic resonance imaging for the detection of breast cancer in young, high-risk women.

BACKGROUND: Evidence-based screening guidelines are needed for women under 40 with a family history of breast cancer, a BRCA1 or BRCA2 mutation, or other risk factors. An accurate assessment of breast cancer risk is required to balance the benefits and risks of surveillance, yet published studies have used narrow risk assessment schemata for enrollment. Breast density limits the sensitivity of film-screen mammography but is not thought to pose a limitation to MRI, however the utility of MRI surveillance has not been specifically examined before in women with dense breasts. Also, all MRI surveillance studies yet reported have used high strength magnets that may not be practical for dedicated imaging in many breast centers. Medium strength 0.5 Tesla MRI may provide an alternative economic option for surveillance. METHODS: We conducted a prospective, nonrandomized pilot study of 30 women age 25-49 years with dense breasts evaluating the addition of 0.5 Tesla MRI to conventional screening. All participants had a high quantitative breast cancer risk, defined as > or = 3.5% over the next 5 years per the Gail or BRCAPRO models, and/or a known BRCA1 or BRCA2 germline mutation. RESULTS: The average age at enrollment was 41.4 years and the average 5-year risk was 4.8%. Twenty-two subjects had BIRADS category 1 or 2 breast MRIs (negative or probably benign), whereas no category 4 or 5 MRIs (possibly or probably malignant) were observed. Eight subjects had BIRADS 3 results, identifying lesions that were "probably benign", yet prompting further evaluation. One of these subjects was diagnosed with a stage T1aN0M0 invasive ductal carcinoma, and later determined to be a BRCA1 mutation carrier. CONCLUSION: Using medium-strength MRI we were able to detect 1 early breast tumor that was mammographically undetectable among 30 young high-risk women with dense breasts. These results support the concept that breast MRI can enhance surveillance for young high-risk women with dense breasts, and further suggest that a medium-strength instrument is sufficient for this application. For the first time, we demonstrate the use of quantitative breast cancer risk assessment via a combination of the Gail and BRCAPRO models for enrollment in a screening trial.

Haploinsufficiency for BRCA1 is associated with normal levels of DNA nucleotide excision repair in breast tissue and blood lymphocytes.

BACKGROUND: Screening mammography has had a positive impact on breast cancer mortality but cannot detect all breast tumors. In a small study, we confirmed that low power magnetic resonance imaging (MRI) could identify mammographically undetectable tumors by applying it to a high risk population. Tumors detected by this new technology could have unique etiologies and/or presentations, and may represent an increasing proportion of clinical practice as new screening methods are validated and applied A very important aspect of this etiology is genomic instability, which is associated with the loss of activity of the breast cancer-predisposing genes BRCA1 and BRCA2. In sporadic breast cancer, however, there is evidence for the involvement of a different pathway of DNA repair, nucleotide excision repair (NER), which remediates lesions that cause a distortion of the DNA helix, including DNA cross-links. CASE PRESENTATION: We describe a breast cancer patient with a mammographically undetectable stage I tumor identified in our MRI screening study. She was originally considered to be at high risk due to the familial occurrence of breast and other types of cancer, and after diagnosis was confirmed as a carrier of a Q1200X mutation in the BRCA1 gene. In vitro analysis of her normal breast tissue showed no differences in growth rate or differentiation potential from disease-free controls. Analysis of cultured blood lymphocyte and breast epithelial cell samples with the unscheduled DNA synthesis assay (UDS) revealed no deficiency in nucleotide excision repair (NER). CONCLUSION: As new breast cancer screening methods become available and cost effective, patients such as this one will constitute an increasing proportion of the incident population, so it is important to determine whether they differ from current patients in any clinically important ways. Despite her status as a BRCA1 mutation carrier, and her mammographically dense breast tissue, we did not find increased cell proliferation or deficient differentiation potential in her breast epithelial cells, which might have contributed to her cancer susceptibility. Although NER deficiency has been demonstrated repeatedly in blood samples from sporadic breast cancer patients, analysis of blood cultured lymphocytes and breast epithelial cells for this patient proves definitively that heterozygosity for inactivation of BRCA1 does not intrinsically confer this type of genetic instability. These data suggest that the mechanism of genomic instability driving the carcinogenic process may be fundamentally different in hereditary and sporadic breast cancer, resulting in different genotoxic susceptibilities, oncogene mutations, and a different molecular pathogenesis.

The GPA in vivo somatic mutation assay.

The glycophorin A (GPA) assay concurrently detects and quantifies two types of erythrocytes with variant phenotypes at the autosomal locus responsible for the polymorphic MN blood group. It uses a pair of allele-specific monoclonal antibodies and flow cytometry to analyze a standard population of 5 million cells efficiently. The two phenotypes detected are simple allele loss and allele loss followed by reduplication of the remaining allele; both are consistent with the mechanisms underlying "loss of heterozygosity" at tumor suppressor genes. The assay is an intermediate biomarker of biological effect, meaning that it integrates both exposure and biological response. It has been applied to populations with a known or suspected genotoxic exposure, to patients with hereditary syndromes causing predisposition to cancer (in which the assay has begun to be moved from validation mode to application), and to patients manifesting a disease endpoint, i.e., cancer.

DNA double-strand break damage and repair assessed by pulsed-field gel electrophoresis.

This assay quantifies the amount of DNA double-strand break (DSB) damage in attached cell populations embedded in agarose and assayed for migratory DNA using pulsed-field gel electrophoresis (PFGE) with ethidium bromide staining. The assay can measure pre-existing damage, as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells are allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in these repaired lanes in comparison with the total amount of DNA fragmentation following determination of a optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.

Molecular analysis of mutations in the human HPRT gene.

The HPRT assay uses incorporation of toxic nucleotide analogs to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosporibosyltransferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.

HPRT gene alterations in umbilical cord blood T-lymphocytes in newborns of mothers exposed to tobacco smoke during pregnancy.

Prenatal exposure to tobacco smoke has been associated with an increased risk of pediatric malignancies, yet the transplacental induction of genetic alterations by tobacco smoke carcinogens and their implication to childhood diseases remain poorly understood. We characterized mutations in the HPRT gene in umbilical cord blood T-lymphocytes of self-reported 103 never-smoking mothers and 104 smoking mothers (54 mothers smoked throughout and 50 mothers quit smoking during pregnancy). The results showed the illegitimate V(D)J recombinase-mediated deletion of HPRT exons 2-3 was the most prominent alteration occurring in 48.2% (26/54) of mutants from neonates of the smoking mothers who smoked during pregnancy, compared with 28.0% (14/50) from those of smoking mothers who quit smoking during pregnancy (p=0.035, Fisher's exact test), 34.9% (36/103) from never-smoking mothers (p=0.08), or 32.7% (50/153) of those of neonates born from the latter two groups of mothers combined (p=0.043). There was no significant difference in the frequency of this deletion between neonates of the never-smoking mothers and the smoking mothers who quit smoking during pregnancy (34.9% versus 28.0%, respectively, p=0.39). The results show an increase in illegitimate V(D)J recombinase-mediated deletion of HPRT exons 2-3 in cord blood T-lymphocytes of newborns of mothers who smoked during pregnancy, compared with the group of mothers who did not smoke during pregnancy, implying an increase in illegitimate V(D)J recombinase-mediated alteration, a genetic recombination event associated with childhood malignancies, may be induced in utero during pregnancy by maternal exposure to tobacco smoke-derived genotoxicants.

Qualitatively and quantitatively similar effects of active and passive maternal tobacco smoke exposure on in utero mutagenesis at the HPRT locus.

BACKGROUND: Induced mutagenesis in utero is likely to have life-long repercussions for the exposed fetus, affecting survival, birth weight and susceptibility to both childhood and adult-onset diseases, such as cancer. In the general population, such exposures are likely to be a consequence of the lifestyle choices of the parents, with exposure to tobacco smoke one of the most pervasive and easily documented. Previous studies attempting to establish a direct link between active smoking and levels of somatic mutation have largely discounted the effects of passive or secondary exposure, and have produced contradictory results. METHODS: Data from three studies of possible smoking effects on in utero mutagenesis at the HPRT locus were compiled and reanalyzed, alone and in combination. Where possible, passive exposure to environmental tobacco smoke was considered as a separate category of exposure, rather than being included in the non-smoking controls. Molecular spectra from these studies were reanalyzed after adjustment for reported mutation frequencies from the individual studies and the entire data set. RESULTS: A series of related studies on mutation at the X-linked HPRT locus in human newborn cord blood samples has led to the novel conclusion that only passive maternal exposure to tobacco mutagens has a significant effect on the developing baby. We performed a pooled analysis of the complete data from these studies, at the levels of both induced mutation frequency and the resulting mutational spectrum. CONCLUSION: Our analysis reveals a more commonsensical, yet no less cautionary result: both active maternal smoking and secondary maternal exposure produce quantitatively and qualitatively indistinguishable increases in fetal HPRT mutation. Further, it appears that this effect is not perceptibly ameliorated if the mother adjusts her behavior (i.e. stops smoking) when pregnancy is confirmed, although this conclusion may also be affected by continued passive exposure.

Are Molecules Involved in Neuritogenesis and Axon Guidance Related to Autism Pathogenesis?

Autism spectrum disorder is a heterogeneous disease, and numerous alterations of gene expression come into play to attempt to explain potential molecular and pathophysiological causes. Abnormalities of brain development and connectivity associated with alterations in cytoskeletal rearrangement, neuritogenesis and elongation of axons and dendrites might represent or contribute to the structural basis of autism pathology. Slit/Robo signaling regulates cytoskeletal remodeling related to axonal and dendritic branching. Components of its signaling pathway (ABL and Cdc42) are suspected to be molecular bases of alterations of normal development. The present review describes the most important mechanisms underlying neuritogenesis, axon pathfinding and the role of GTPases in neurite outgrowth, with special emphasis on alterations associated with autism spectrum disorders. On the basis of analysis of publicly available microarray data, potential biomarkers of autism are discussed.

Regulation and disregulation of mammalian nucleotide excision repair: a pathway to nongermline breast carcinogenesis.

Nucleotide excision repair (NER) is an important modulator of disease, especially in constitutive deficiencies such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation in NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals and oxidative stress. Because NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NER pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. As NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic modulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially influences response to therapy. In this study, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer.