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A K Godwin

Publications and source records attributed to A K Godwin.

At least 55 records · Page 3Linked to original sources
PubMed1996

Alternative splicing and differential expression of DT-diaphorase transcripts in human colon tumors and in peripheral mononuclear cells in response to mitomycin C treatment.

The two-electron bioreductive enzyme DT-diaphorase catalyzes the metabolism of quinones. The existence of several distinct sizes of DT-diaphorase mRNA transcripts has been observed in human tissues. One of these, an alternatively spliced mRNA that lacks exon 4, has been recently found to be expressed at levels comparable to those of the full-length mRNA. The protein encoded by the mRNA lacking exon 4 has minimal catalytic activity, consistent with the elimination of the quinone-binding site coded for by this exon. We have pursued a number of approaches to examine the significance of this splice variant. We identified a similar truncated transcript in a human HepG2 cDNA library. To determine the frequency of expression of this form of DT-diaphorase in the general population, we examined mRNA obtained from the peripheral mononuclear cells of 16 patients and found substantial interindividual variability in the patterns of transcript expression. Following treatment of these 16 patients with 20 mg/m2 mitomycin C (MMC), the induction of DT-diaphorase transcripts was demonstrated. In most patients, expression of the variant transcript (lacking exon 4) remained constant, while that of the full-length mRNA was elevated. The extent of induction also showed interindividual variability. In one patient, while both transcripts were present at baseline, expression of the variant transcript disappeared almost completely after MMC treatment. To analyze these events under more controlled conditions, we examined the effects of MMC treatment on two human colon tumor cell lines. MMC treatment induced expression of the full-length mRNA but did not influence the abundance of the variant transcript. We then performed single-strand conformational polymorphism analysis of genomic DNA from the 16 patients to investigate the potential role of cis-acting factors in the variable splicing responses. Two patients demonstrated sequence differences in the region spanning exon 4, but in neither was the change in a region critical to splicing regulation. These data demonstrate that the expression of DT-diaphorase in hyman cells is polymorphic, and that the levels of individual transcripts can be regulated by exogenous factors. The findings support a role for alternative splicing in the control of DT-diaphorase gene expression.

Alternative Splicing↗
PubMed1996

A Grb2-associated docking protein in EGF- and insulin-receptor signalling.

The protein Grb2 plays a central role in signalling by receptor protein-tyrosine kinases, where its SH2 domain binds to the receptor and its two SH3 domains link to effectors. One target effector is Sos, so Grb2 links receptor protein-tyrosine kinases with the Ras signalling pathway. The SH3 domains can also couple to other signalling proteins, including Vav, c-Abl and dynamin. We have identified several bands in glial and medulloblastoma tumours that are recognized by Grb2 but these did not correspond to any known protein. Here we use recombinant Grb2 to isolate a complementary DNA called Gab1 (for Grb2-associated binder-1). Gab1 shares amino-acid homology and several structural features with IRS-1 (insulin-receptor substrate-1; refs 6,7), is a substrate of the EGF and insulin receptors, and can act as a docking protein for several SH2-containing proteins. Over-expression of Gab1 enhances cell growth and results in transformation. We conclude that Gab1 is a new protein in EGF and insulin receptor signalling which could integrate signals from different systems.

3T3 Cells↗
PubMed1996

Variability in loss of constitutional heterozygosity across loci and among individuals: association with candidate genes in ductal breast carcinoma.

Genes involved in the metabolic activation or detoxification of environmental carcinogens may contribute to breast cancer susceptibility by influencing rates of somatic mutation. To examine this hypothesis, we studied the association between loss of constitutional heterozygosity (LOH) in ductal breast tumors and allelic variability in genes that regulate the metabolism of environmental carcinogens. LOH was measured by typing the tumor and normal tissue of 28 breast cancer cases at 33 chromosomal loci by using highly polymorphic tetranucleotide repeat markers. Genotypes in non-tumor tissue were also measured at the cytochrome P450 1A1 (CYP1A1), cytochrome P450 2D6 (CYP2D6), glutathione-s-transferase mu (GSTM), epoxide hydrolase (EH), and NAD(P)H:quinone oxidoreductase (NQO1) loci. The observed proportion of LOH was 11% overall and ranged from 0% to 37% across loci. LOH greater than 20% was observed on chromosomes 1p, 2p, 10q, 11q, 17p, and 18q. The observed proportion of LOH ranged from 0% to 67% among individuals. An elevated proportion of LOH was observed for genotypes at CYP2D6 (17% for the 1/1 and 1/2 genotypes vs 8% for the 2/ 2 genotype), NQO1 (13% for the 1/2 and 2/2 genotypes vs 8% for the 1/1 genotype), and GSTM (15% for the null genotype vs 7% for the wild-type genotype). No elevated proportion of LOH was observed for genotypes at CYP1A1 (12% for the 1/2 genotype vs 10% for the 1/1 genotype) or EH (11% for the 1/1 genotype vs 10% for the 1/2 genotype). There was no correlation of LOH with any other tumor characteristic such as estrogen- or progesterone-receptor status or number of positive lymph nodes. These results suggest that the proportion of LOH varies substantially across loci and among individuals. Interindividual variability in LOH may thus be explained in part by genes that regulate the metabolism of environmental carcinogens.

Adult↗
PubMed1996

BRCA2 germline mutations in male breast cancer cases and breast cancer families.

The breast cancer susceptibility gene, BRCA2 on chromosome 13q12-13, was recently isolated. Mutations in BRCA2 are thought to account for as much as 35% of all inherited breast cancer as wall as a proportion of inherited ovarian cancer. Many BRCA2-linked families also contain cases of male breast cancer. We have analysed germline DNA from 50 males with breast cancer (unselected for family history) and 26 individuals from site-specific female breast and breast-ovarian cancer families for mutations in BRCA2. All 17 breast-ovarian cancer families have been screened for BRCA1 coding region mutations and none were detected. Conformation-sensitive gel electrophoresis (CSGE) analysis of PCR-amplified DNA followed by direct sequencing was used to detect sequence variants. Three of eleven individuals carry the same mutation, all are of Ashkenazi Jewish descent, supporting the observation by Neuhausen et al. in this issue that there is a common mutation in this population. Eleven truncating mutations and nine polymorphisms were identified -- all were coding region variants. No loss-of-transcript mutations were identified in the sixteen samples for which this analysis was possible. Seven of the nine disease-associated mutations were detected in the 50 men with breast cancers; for thus in our series, BRCA2 mutations account for 14% of male breast cancer, all but one of which had a family history of male and/or female breast cancer.

BRCA2 Protein↗
PubMed1996

Two distinct origins of a common BRCA1 mutation in breast-ovarian cancer families: a genetic study of 15 185delAG-mutation kindreds.

We screened 163 women from breast-ovarian cancer-prone families, as well as 178 individuals affected with breast and/or ovarian cancer but unselected for family history, for germ-line mutations in exon 2 of BRCA1, by SSCP analysis and direct sequencing. A total of 25 mutations were detected. Thirteen of 64 Jewish Ashkenazi women and 2 non-Jewish individuals were found to possess the 185delAG mutation. Haplotype data for all 15 individuals, with markers intragenic to BRCA1, suggest that the Jewish Ashkenazi individuals share a common ancestry that is distinct from the lineage shared by the other two women. These data provide the first evidence of two distinct lines of transmission for the 185delAG mutation, only one of which has its origins in the Jewish Ashkenazi population. Our screening also uncovered 10 affected individuals with an 11-bp deletion at nucleotide 188 of BRCA1 (188del11), 4 of whom are Ashkenazi Jews. This is only the third reported mutation detected within the Jewish Ashkenazi population and may represent the second most common alteration in BRCA1 found in Ashkenazi Jews in the United States. The observed overrepresentation of specific mutations within a subgroup of the general population may eventually contribute to the development of inexpensive and routine tests for BRCA1 mutations, as well as to the elucidation of other contributory factors (e.g., diet, environment, and chemical exposures) that may play a key role in cancer initiation and development. The implications of the mutational data, as well as the role that founder effect, demographic history, and penetrance play in the resulting observed phenomena, are discussed.

Adult↗
PubMed1995

Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors.

The epidermal growth factor receptor has received much interest as a target for various antineoplastic agents, but a complication is that many normal tissues also express this receptor. We have previously identified in human glial tumors an 801-bp in-frame deletion within the epidermal growth factor receptor gene that created a novel epitope at the junction. By using Western blot assays with a mutant-specific antibody as a rapid and sensitive means for detecting this alteration in primary human tumors, it was found that 57% (26 of 46) of high-grade and 86% (6 of 7) of low-grade glial tumors, but not normal brain, express this protein. This altered receptor was also present in 66% (4 of 6) of pediatric gliomas and 86% (6 of 7) of medulloblastomas, 78% (21 of 27) of breast carcinomas, and 73% (24 of 32) of ovarian carcinomas. The fact that this receptor is frequently found in tumors but not in normal tissue makes it an attractive candidate for various antitumor strategies.

Antibodies, Monoclonal↗
PubMed1995

Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines.

We have shown previously that tumor cell resistance to cisplatin is associated with elevated intracellular levels of glutathione, which is accomplished at least in part by increased expression of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS). To investigate the mechanism by which gamma-GCS expression is elevated, we have examined four related human ovarian cancer cell lines with increasing cisplatin resistance. Relative amounts of steady-state gamma-GCS mRNA in CP70, C30, and C200 were 4.8, 6.0, and 10.6, respectively, compared to the parental A2780 cell line, and a proportional increase in the transcriptional rate but not RNA stability was demonstrated. In contrast, no increase in mRNA for the gamma-GCS light subunit was found. To determine the mechanism of upregulation of this mRNA, we have cloned the promoter of the gene that encodes the heavy subunit of gamma-GCS. This region contains AP-1, NF-kappa B, XRE, AP-2, EpRE, CAAT, and TATA box elements upstream of the transcription initiation site and two MREs between this site and the start codon for the protein. Using gel mobility shift assays, we have found nuclear extract binding activity to the AP-1 response element to be closely associated with the level of gamma-GCS gene expression. A supershift assay showed that the AP-1 DNA-binding complexes are predominantly formed by dimers of JUN family members. Consistent with this finding, the expression of c-JUN was found to be elevated in the resistant cells. In contrast to AP-1 binding, AP-2 and NF-kappa B binding were inversely related to resistance. Furthermore, we have examined a partial revertant of the C200-resistant cells, which shows lower glutathione levels, and found decreased gamma-GCS expression associated with decreased AP-1 binding activity.

Antineoplastic Agents↗
PubMed1995

Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas.

The AKT2 gene is one of the human homologues of v-akt, the transduced oncogene of the AKT8 virus, which induces lymphomas in mice. In previous studies, AKT2, which codes for a serine-threonine protein kinase, was shown to be amplified and overexpressed in some human ovarian carcinoma cell lines and amplified in primary tumors of the ovary. To confirm and extend these findings, we conducted a large-scale, multicenter study of AKT2 alterations in ovarian and breast cancer. Southern-blot analysis demonstrated AKT2 amplification in 16 of 132 (12.1%) ovarian carcinomas and in 3 of 106 (2.8%) breast carcinomas. No AKT2 alteration was detected in 24 benign or borderline tumors. Northern-blot analysis revealed overexpression of AKT2 in 3 of 25 fresh ovarian carcinomas which were negative for AKT2 amplification. The difference in the incidence of AKT2 alterations in ovarian and breast cancer suggests a specific role for this gene in ovarian oncogenesis. No significant association was found between AKT2 amplification and amplification of the proto-oncogenes MYC and ERBB2, suggesting that amplification of AKT2 defines an independent subset of breast and ovarian cancers. Ovarian cancer patients with AKT2 alterations appear to have a poor prognosis. Amplification of AKT2 was especially frequent in undifferentiated tumors (4 of 8, p = 0.019), suggesting that AKT2 alterations may be associated with tumor aggressiveness.

Adenocarcinoma↗
PubMed1995

Characterization of chromosome 9 in human ovarian neoplasia identifies frequent genetic imbalance on 9q and rare alterations involving 9p, including CDKN2.

We have examined 41 forms of ovarian cancer for genetic alterations on chromosome 9 using a combination of five RFLP DNA probes and 15 simple tandem repeat polymorphisms. Genetic imbalance (i.e., loss of heterozygosity, microsatellite instability, amplification) for 1 or more informative markers on chromosome 9 was observed in 66% (27 of 41) of our tumor panel. Genetic imbalance was observed on 9q in 59% (24 of 41) of tumors informative for at least one locus. In contrast, only 13% (5 of 40) of informative tumors demonstrated a genetic alteration involving 9p. Furthermore, allelic loss on 9q was more common in late stage tumors (63%, 17 of 27) and poorly differentiated tumors (75%, 15 of 20) as compared to benign and early stage tumors (30%, 3 of 10). Evaluation of 15 tumors showing limited regions of genetic imbalance has identified 2 candidate tumor suppressor regions on 9q and 1 on 9p. Interestingly, the regions defined to 9p21-p24, 9q31, and 9q32-q34 all overlap with several known disease loci. In this aspect, the potential role of the CDKN2 gene at 9p21-p22 in ovarian carcinogenesis was assessed in an extended panel of ovarian tumors, 11 human ovarian carcinoma cell lines, and 1 cervical tumor cell line. With the use of comparative multiplex PCR, homozygous deletions were detected in 16 of 115 (14%) fresh tumors and 3 of 12 cancer cell lines. For those tumors demonstrating allelic loss for markers on 9p no somatic mutations were observed in the retained allele of CDKN2, as determined by single-strand conformation polymorphism analysis, but a mutation was observed in an additional cell line. Furthermore, CDKN2 mRNA levels were similar in the 9 cancer cell lines that retain CDKN2, as compared to normal human ovarian surface epithelial cell lines. Overall, our results suggest the potential involvement of a gene or genes on chromosome 9q and de-emphasize a significant role for the CDKN2 gene on 9p in the initiation and progression of ovarian cancer.

Adenocarcinoma, Mucinous↗
PubMed1995

Expression of two mucin antigens in cultured human ovarian surface epithelium: influence of a family history of ovarian cancer.

OBJECTIVE: The genetic changes in hereditary ovarian cancer syndromes suggest that the phenotype of ovarian surface epithelium in women with familial ovarian cancer might be altered. To test this hypothesis, we compared two tumor markers, CA 125 and 2G3, in cultures of overtly normal epithelium from patients with and without familial ovarian cancer. STUDY DESIGN: Surface epithelia from 18 patients with no family history of ovarian cancer, five with family histories that were insufficient to be classified as familial, and seven with strong family histories were examined by immunofluorescence, immunocytochemistry, and radioimmunoassay. The influence of cell density, morphologic features, propagation in culture, and immortalization with SV40 on the expression of the markers was investigated. RESULTS: CA 125 occurred in cells with epithelial rather than atypical morphologic features. In cultures with no family history and minor family history, CA 125 was present in up to 45% cells in passage 1 but in only < 5% cells in 14 of 16 cultures by passages 3 to 4. In contrast, nine of 10 cultures with family history retained > 5% CA 125-positive cells in passages 3 to 4. This prolonged presence of CA 125 correlated with a persisting epithelial phenotype, whereas most cells with no family history and minor family history became atypical by passage 3. Immortalization eliminated CA 125 in all three types of cells. 2G3 bound to few cells in low passage, independent of family history and morphologic features. The proportion of 2G3-expressing cells increased significantly with immortalization in all cultures, independent of family history. Ovarian carcinoma lines expressed both markers. CONCLUSION: In cultures of ovarian surface epithelium 2G3 expression increases with immortalization, whereas CA 125 is lost with immortalization but correlates with epithelial cell morphologic features and with family history. The results suggest that there may be phenotypic changes in overtly normal ovarian surface epithelium of women with family histories of ovarian cancer.

Antigens, Neoplasm↗
PubMed1995

Retroviral-like sequences specifically expressed in the rat ovary detect genetic differences between normal and transformed rat ovarian surface epithelial cells.

We have identified a repetitive DNA element in the rat genome that we demonstrate to be suitable to detect molecular genetic differences between normal and malignantly transformed rat ovarian surface epithelial cells by genome scanning. With fluorescence in situ hybridization, we show that these elements are widely distributed in the rat genome, and that a member of this family is present within a homogeneously staining chromosomal region of tumorigenic rat ovarian surface epithelial cells. The homogeneously staining chromosomal region infers the presence of an amplified DNA sequence in the tumor cells, and we provide molecular evidence for an amplicon in this DNA by genome scanning using a probe related to these elements. Sequence analysis revealed that these elements have the structural features of retroviral DNA. We show that they are transcriptionally active in the rat ovary, but not in a wide range of other normal rat tissues. In situ hybridization to ovarian tissue sections revealed that the elements are expressed in granulosa and theca interna cells and in the surface epithelial cells adjacent to preovulatory follicles. Based on this property, we refer to these retroviral-like elements as ovary-specific transcribed sequences. Partial sequence analysis of multiple members of this family and the range in transcript sizes strongly suggest that multiple members of this retrovirus-like family are transcriptionally active. We speculate that binding by one or a combination of ovary-specific transcription factors to regulatory sequences within the genomic ovary-specific transcribed sequences units contributes to the tissue specificity of their expression.

Animals↗
PubMed1994

Relationship between platinum-DNA adduct formation and removal and cisplatin cytotoxicity in cisplatin-sensitive and -resistant human ovarian cancer cells.

We examined several aspects of platinum-DNA adduct formation and repair in cisplatin-sensitive and -resistant human ovarian cancer cell lines. The formation of cisplatin-interstrand crosslinks (ICLs) was measured in five DNA sequences by renaturing agarose gel electrophoresis. There were considerable differences (up to 4-fold) in ICL levels in these DNA sequences following a 4-h incubation with cisplatin; however, the pattern of ICL formation did not depend on whether the region was transcriptionally active or gene encoding. Incubation of purified DNA with cisplatin yielded an ICL pattern with considerably less variability between the regions examined. Cisplatin ICL and total DNA platination levels were significantly higher (up to 20- and 40-fold, respectively) in cisplatin-resistant cell lines as compared to the parental, cisplatin-sensitive cell line at equivalent levels of cisplatin cytotoxicity. Under cisplatin exposure conditions which yielded similar initial levels of sequence-specific ICLs, the cisplatin-resistant cells removed up to 2.5 times more ICLs by 12-h posttreatment than the parental cell line. Increased removal of the individual platinum-deoxyribonucleosides of platinum-DNA adducts was also observed in the highly resistant C200 cell line as determined by high performance liquid chromatography separation and quantitation by atomic absorption spectrometry. These results indicate that DNA repair contributes significantly to cisplatin resistance and that increased DNA-damage tolerance may also be a component of the resistance phenotype in this model system.

Cisplatin↗
PubMed1994

Effects of hypoxia on detoxicating enzyme activity and expression in HT29 colon adenocarcinoma cells.

Resistance of hypoxic tumor cells to ionizing radiation and cytotoxic drugs has been attributed to changes in the reactivity and/or the half-times of reactive species in the altered redox environment. Exposure of eukaryotic cells to such hypoxic conditions results in the induction of the synthesis of several unrelated proteins. To investigate further the phenomenon of hypoxic cell resistance to cytotoxic drugs, we examined the effects of hypoxia on the expression of a group of enzymes involved in drug metabolism. Exposure of HT29 colon carcinoma cells to hypoxia resulted in a marked increase in the activity of DT-diaphorase and in glutathione content. The activity of glutathione transferase was not increased by this treatment. The response was proportional to the duration of hypoxia. After the cells were exposed to hypoxic conditions for 8 h, followed by restoration of an oxic environment, the elevation in enzyme activity and glutathione content reached a peak at 48 h (40 h after the restoration of an oxic environment) and returned to baseline at 72 h. Elevation of steady-state levels of DT-diaphorase and gamma-glutamylcysteine synthetase mRNA followed a similar time course, with > 10-fold increases over oxic cells at 24 h. The elevation of DT-diaphorase mRNA content was found to result both from transcriptional induction and from increased message stability. The magnitude and persistence of elevated detoxicating enzyme activity following a relatively short hypoxic exposure followed by reoxygenation suggest a novel potential mechanism of resistance to cytotoxic drugs in hypoxic tumors.

Adenocarcinoma↗
PubMed1994

Spontaneous transformation of rat ovarian surface epithelial cells results in well to poorly differentiated tumors with a parallel range of cytogenetic complexity.

We previously used rat ovarian surface epithelial cells subjected to repetitious growth in vitro to provide experimental evidence in support of a role for incessant ovulation in the etiology of ovarian cancer. We have now initiated a series of 30 independent rat ovarian surface epithelial cell lines. This report describes findings in eight of the cell lines that, based on tumor formation in athymic mice, have undergone malignant transformation. Each of the tumors exhibited chromosomal alterations. Two well to moderately differential tumors had only one or two cytogenetic changes, and they had in common the presence of numerical gains. Each of five poorly differentiated tumors had complex karyotypes with three to eight clonal aberrations, prominent among them being unbalanced rearrangements and numerical losses. Several poorly differentiated tumors also had marker chromosomes, double minutes, or homogeneously staining regions. These findings demonstrate that the malignant tumors produced by spontaneously transformed rat ovarian surface epithelial cell lines range in degree of differentiation, which is paralleled by the cytogenetic complexity. Thus, this model system may fill an important void in future efforts to define the genetic basis of common epithelial tumors of the ovary and many features characteristic of these neoplasms.

Adenocarcinoma↗
PubMed1994

Role of platinum-DNA adduct formation and removal in cisplatin resistance in human ovarian cancer cell lines.

A series of cisplatin-resistant cell lines were used to examine the formation and removal of platinum-DNA adducts from the overall genome and the formation and removal of cisplatin-interstrand cross-links from specific genomic regions. Cisplatin accumulation and DNA platination levels, which correlated linearly, were similar in three of the resistant cell lines despite differences in their primary cisplatin resistance. Increased platinum removal from total genomic DNA was found to be associated with increased resistance. Interstrand cross-link levels were found to be 2- to 4-fold lower in the 28S ribosomal RNA gene and a non-coding genomic region of the resistant cell lines as compared with the parental A2780 cell line. In addition, 1.2- to 2.7-fold more cross-links were formed in the non-coding region than in the ribosomal RNA gene in all of the cell lines. Interstrand cross-links were removed more rapidly from both regions of the highly cisplatin-resistant C80 and C200 cells and from the ribosomal RNA gene only in the cell lines of lower resistance. The results support a role for DNA repair and alterations in interstrand cross-link formation in cisplatin resistance and provide evidence for heterogeneous interstrand cross-link formation in the genome.

Cisplatin↗