Biomedical subjects

J R Marks

Publications and source records attributed to J R Marks.

At least 37 records · Page 2Linked to original sources

PubMed1996

Inhibition of the erbB-2 tyrosine kinase receptor in breast cancer cells by phosphoromonothioate and phosphorodithioate antisense oligonucleotides.

Antisense activity against erbB-2 of a variety of sulfur-modified oligonucleotides was examined in a breast cancer cell line which overexpresses this oncogene. Using a 15 base anti-erbB-2 sequence previously shown to be effective, various backbone configurations containing phosphoromonothioate or phosphorodithioate linkages were evaluated for antisense activity by a two-color flow cytometric assay. This sequence was effective in inhibiting the production of erbB-2 protein when it was configured as a monothioate at each linkage and as an alternating dithioate/phosphodiester. Both of these compounds were also able to specifically inhibit erbB-2 mRNA expression, indicative of RNase H-mediated activity. The same sequence protected by either three dithioate or three monothioate linkages at each end was ineffective as an antisense reagent, suggesting that endonuclease activity is a significant determinant of the stability of oligonucleotides. Finally, the erbB-2 sequence target was shifted in an effort to improve antisense activity. A new lead sequence was identified that was significantly more effective in inhibiting erbB-2 protein levels and retained activity at lower concentrations.

Base Sequence↗

PubMed1996

Cell cycle control of BRCA2.

Identifying the conditions and kinetics of the induction of BRCA2 gene expression may implicate roles for the function of the tumor suppressor gene. In this study, expression of BRCA2 mRNA is shown to be regulated by the cell cycle and associated with proliferation in normal and tumor-derived breast epithelial cells. Cells arrested in G(0) or early G1 contained low levels of BRCA2 mRNA. After release into a proliferating state, cells produced maximum levels of BRCA2 mRNA in late G1 and the S-phase. Similar cell cycle control of BRCA2 was observed in fractions of exponentially growing cells isolated by centrifugal elutriation. Expression of BRCA2 was shown to be independent of bulk DNA synthesis. In addition, the kinetics of BRCA2 mRNA up-regulation appeared to be similar to those of BRCA1, suggesting that the two genes could be commonly controlled. These results imply that these two tumor suppressor genes are utilized during growth and may have a protective role in cellular proliferation.

Adult↗

PubMed1996

M6P/IGF2 receptor: a candidate breast tumor suppressor gene.

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r) functions in the activation of TGFbeta, a potent growth inhibitor for most cell types, the degradation of the mitogen, IGF2, and the intracellular trafficking of lysosomal enzymes. We have found its expression to be significantly reduced in both rat and human hepatocellular carcinomas (HCCs) and recently reported loss of heterozygosity (LOH) at this locus with mutations in the remaining allele in human liver tumors. Using the polymerase chain reaction, we utilized two polymorphisms in the 3' untranslated region of M6P/IGF2r to screen breast tumors for LOH. Forty of 62 (65%) patients were informative (heterozygous) and 12/40 (30%) breast tumors had LOH; 5/19 (26%) carcinomas in situ (CIS) and 7/21 (33%) invasive carcinomas. To investigate the early molecular genetic events in breast carcinogenesis, we screened the CIS with LOH for mutations. In 2/5 (40%) of these tumors, missense mutations were found in the remaining allele that gave rise to significant amino acid substitutions. These findings provide evidence that M6P/IGF2r allelic loss is an early event in the etiology of breast cancer, that this gene functions as a tumor suppressor gene in the breast.

Amino Acid Sequence↗

PubMed1996

Microsomal epoxide hydrolase polymorphism as a risk factor for ovarian cancer.

Microsomal epoxide hydrolase (EPHX) is one of many enzymes involved in the metabolism of endogenous and exogenous toxicants. Polymorphic forms of the human EPHX gene have been described that vary in enzymatic activity, and one, Tyr113His, has been associated with hepatocellular carcinoma susceptibility. We demonstrated that EPHX was highly expressed in the human ovary, and investigated whether specific EPHX genotypes are associated with ovarian cancer susceptibility. Seventy-three Caucasian patients with ovarian cancer and 75 Caucasian-female controls without cancer were genotyped for the Tyr113His polymorphism by a polymerase chain reaction-restriction fragment length polymorphism assay. The frequency of the homozygous high-activity genotype was 41% in the control population and 64% in the ovarian cancer patients. The odds ratio for ovarian cancer with this genotype was 2.6 (95% confidence interval 1.3, 5.0; P < 0.01). The increased ovarian cancer risk associated with the high-activity genotype could reflect differences in metabolic activation of endogenous or exogenous carcinogens.

Blotting, Northern↗

PubMed1996

p53 overexpression in advanced-stage endometrial adenocarcinoma.

OBJECTIVES: Mutation and overexpression of the p53 tumor suppressor gene in endometrial cancers are associated with advanced stage and poor survival. We sought to determine whether p53 overexpression is an independent variable predictive of poor prognosis in advanced endometrial adenocarcinomas. STUDY DESIGN: Immunohistochemical evaluation was used to examine p53 expression in paraffin blocks from 179 endometrial adenocarcinomas. RESULTS: p53 overexpression was seen in 35% of cancers and was associated with higher stage (p = 0.004), black race (p < 0.001), higher grade (p = 0.02), lack of hormone replacement (p = 0.04), and older age (p = 0.05). In addition to a higher frequency of p53 overexpression (57% vs 26%), black women had a lower survival rate than white women (p = 0.001), but overexpression was associated with poor survival in both races. After we corrected for hormone use, multivariate analysis revealed that older age (p < 0.001), higher stage (p < 0.001), higher grade (p = 0.01), and p53 overexpression (p = 0.04) were predictive of poor survival. CONCLUSIONS: Overexpression of p53 in advanced-stage endometrial cancers is an independent variable that is associated with poor survival, occurs more frequently in black women, and may contribute to the racial disparity in survival.

Adenocarcinoma↗

PubMed1996

BRCA2 mutations in primary breast and ovarian cancers.

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

Aged↗

PubMed1996

Role of BRCA1 mutation screening in the management of familial ovarian cancer.

Families with multiple cases of ovarian cancer have long been observed, and in the past prophylactic oophorectomy has been advocated for women with a history of ovarian cancer in two first-degree relatives. It is now thought that > 90% of familial ovarian cancer is due to inherited mutations in the BRCA1 breast-ovarian cancer susceptibility gene on chromosome 17q. BRCA1 testing is being performed in several academic medical centers on a research basis and is also now commercially available. With the ability to identify inherited mutations in BRCA1, prophylactic oophorectomy and other interventions intended to decrease cancer mortality can be offered specifically to women who carry a mutation, but the optimal strategy for decreasing cancer mortality in BRCA1 families has not yet been determined. To facilitate further clinical and basic research in this field, our group and others have established multidisciplinary hereditary breast-ovarian cancer clinics that offer a wide range of services including BRCA1 testing, genetic counseling, and cancer prevention and treatment.

Chromosomes, Human, Pair 17↗

PubMed1996

BRCA1 expression is induced before DNA synthesis in both normal and tumor-derived breast cells.

Insight into the function of the BRCA1 tumor suppressor gene may be gained by studying its regulation. In this study, the expression of BRCA1 was examined as a function of the cell cycle in normal and tumor-derived breast epithelial cells. Cells arrested in G(zero) or early in G1 contained low levels of BRCA1 mRNA. After release, populations of cells reached maximal levels of BRCA1 in late G1 and S phase. Induction of BRCA1 was shown to occur before the onset of DNA synthesis by synchronizing cells at the G1-S boundary. Levels of the BRCA1 protein were regulated in a similar manner. No difference was observed between primary cultures of normal mammary epithelial cells and immortalized tumor-derived cell lines. These results suggest that BRCA1 may function at the G1-S checkpoint.

Adult↗

PubMed1996

Tissue transglutaminase expression in human breast cancer.

Tissue transglutaminase (tTG) is postulated to play a role in apoptosis, cell adhesion, metastasis, and extracellular matrix (ECM) assembly. In this study, the distribution and expression of tissue transglutaminase was investigated in normal human mammary tissue and in intraductal and invasive human breast cancer by immunohistochemistry and in situ hybridization. Frozen and formalin-fixed paraffin-embedded sections of normal, intraductal, and invasive human breast carcinoma were examined with an avidin-biotin complex immunoperoxidase method for tTG antigen and by in situ hybridization to determine the cell types expressing tTG mRNA. The expression of tTG in normal and malignant mammary epithelium in culture was evaluated by quantitative immunoblot analysis. Low-level expression of tTG was found in normal tissues with the antigen located in the ECM surrounding the ducts and in the endothelium. In intraductal cancer, there was a marked increased expression of the tTG antigen, and the increased staining was found in the ECM and was also localized in a distinct pattern at the boundary between the in situ tumor cells and the normal tissue. Further immunohistochemical analysis revealed that the cells in this boundary also stained for the endothelial cell markers CD31, CD34, and von Willebrand factor. In invasive tumors, the tTG antigen was no longer localized to the normal tissue/tumor boundary but dispersed around the tumor cells. In situ hybridization studies revealed three distinct compartments of tTG synthesis: (a) tumor cells, (b) endothelial cells, and (c) stromal cells. In addition, normal and malignant epithelial cells in culture expressed variable amounts of tTG, and the expression of tTG in these epithelial cells was at least 17-fold less than endothelial cells. The up-regulation of tTG in intraductal and invasive human breast cancer and its localization to the ECM and neovasculature suggest that tTG may regulate tumor growth and metastasis.

Breast Neoplasms↗

PubMed1996

Relationship between p21 expression and mutation of the p53 tumor suppressor gene in normal and malignant ovarian epithelial cells.

In many cell types, p53-mediated growth inhibition is dependent on induction of p21, which is an inhibitor of cyclin-dependent kinases that are required for cell cycle progression. Failure of mutant p53 proteins to transactivate p21 may lead to uncontrolled proliferation. Because many ovarian cancers have mutations in the p53 gene, we examined p21 levels in normal and malignant ovarian epithelial cells to determine whether p21 expression is dependent on wild-type p53. Normal ovarian epithelial cells and two ovarian cancer cell lines with wild-type p53 expressed readily detectable levels of p21, whereas in p53 null and mutant cell lines, expression of p21 was diminished strikingly. A correlation between the status of the p53 gene and p21 expression also was noted in 23 primary epithelial ovarian cancers. Normal levels of p21 RNA were seen in 4/7 (57%) cancers with wild-type p53, whereas 14/16 (88%) cancers with mutant p53 had reduced p21 expression (P < 0.05). In addition, we found that lambda-irradiation of normal and malignant ovarian epithelial cells with wild-type, but not mutant, p53 resulted in induction of p21. These data are suggestive that induction of p21 is a feature of p53-mediated growth inhibition in normal ovarian epithelial cells. Conversely, mutation of the p53 gene in ovarian cancers usually is associated with decreased p21 expression. The lack of an absolute correlation between p21 expression and the status of the p53 gene in ovarian cancers is consistent with other studies that have suggested that p21 may also be regulated by p53-independent pathways.

Blotting, Northern↗

PubMed1995

Identification of a new subclass of Alu DNA repeats which can function as estrogen receptor-dependent transcriptional enhancers.

We have utilized a genetic selection system in yeast to identify novel estrogen-responsive genes within the human genome and to define the sequences in the BRCA-1 gene responsible for its estrogen responsiveness. This approach led to the identification of a new subclass within the Alu family of DNA repeats which have diverged from known Alu sequences and have acquired the ability to function as estrogen receptor-dependent enhancers. Importantly, these new elements confer receptor-dependent estrogen responsiveness to a heterologous promoter when assayed in mammalian cells. This transcriptional activity can be attenuated by the addition of either of three different classes of estrogen receptor antagonists, indicating that these elements function as classical estrogen receptor-dependent enhancers. Furthermore, this enhancer activity is restricted to a specific subset of DNA repeats because consensus Alu elements of four major subfamilies do not respond to the estrogen receptor. Previously, most Alu sequences have been considered to be functionally inert. However, this work provides strong evidence that a significant subset can confer estrogen responsiveness upon a promoter within which they are located. Clearly, Alu sequences must now be considered as important contributors to the regulation of gene transcription in estrogen receptor-containing cells.

Animals↗

PubMed1995

Antisense DNA downregulation of the ERBB2 oncogene measured by a flow cytometric assay.

A causal role has been inferred for ERBB2 overexpression in the etiology of breast cancer and other epithelial malignancies. The development of therapeutics that inhibit this tyrosine kinase cell surface receptor remains a high priority. This report describes the specific downregulation of ERBB2 protein and mRNA in the breast cancer cell line SK-BR-3 by using antisense DNA phosphorothioates. An approach was developed to examine antisense effects which allows simultaneous measurements of antisense dose and gene specific regulation on a per cell basis. A fluorescein isothiocyanate end-labeled tracer oligonucleotide was codelivered with antisense DNA followed by immunofluorescent staining for ERBB2 protein expression. Two-color flow cytometry measured the amount of both intracellular oligonucleotide and ERBB2 protein. In addition, populations of cells that received various doses of nucleic acids were physically separated and studied. In any given transfection, a 100-fold variation in oligonucleotide dosage was found. ERBB2 protein expression was reduced greater than 50%, but only in cells within a relatively narrow uptake range. Steady-state ERBB2 mRNA levels were selectively diminished, indicating a specific antisense effect. Cells receiving the optimal antisense dose were sorted and analyzed for cell cycle changes. After 2 days of ERBB2 suppression, breast cancer cells showed an accumulation in the G1 phase of the cell cycle.

Base Sequence↗

PubMed1995

A prognostic model of recurrence and death in stage I non-small cell lung cancer utilizing presentation, histopathology, and oncoprotein expression.

In order to construct a multivariate model for predicting early recurrence and cancer death for patients with stage I non-small cell lung cancer, 271 consecutive patients (mean age, 63 +/- 8 years) who were diagnosed, treated, and followed at one institution were studied. All patients were clinical stage I with head and chest/abdominal computed tomograms and radionuclide bone scans without evidence of metastatic disease. Pathological material after resection was reviewed to verify histological staging. Follow-up documented the time and location of any recurrence, was a median 56 months in duration, and was complete in all cases. Data recorded included age, sex, smoking history, presenting symptoms, pathological description, and oncoprotein staining for erbB-2 (HER-2/neu), p53, and KI-67 proliferation protein. Immunohistochemistry of oncogene expression was performed on two separate archived paraffin tumor blocks for each patient, with normal lung as control. All analyses were blinded and included Kaplan-Meier survival estimates with Cox proportional hazards regression modeling. Data, including immunohistochemistry, were complete for all 271 patients. Actual 5-year survival was 63% and actuarial 10-year survival was 58%. Significant univariate predictors (P < 0.05) of early recurrence and cancer-death were: male sex; the presence of symptoms; chest pain; type of cough; hemoptysis; tumor size > 3 cm diameter (T2); poor differentiation; vascular invasion; erbB-2 expression; p53 expression; and a higher KI-67 proliferation index (> 5%). An additive oncogene expression curve demonstrated a 5-year survival of 72% for 136 patients without p53 or erbB-2, 58% for 108 patients who expressed either oncogene, and 38% for 27 who expressed both (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1995

Maintenance of DNA content and erbB-2 alterations in intraductal and invasive phases of mammary cancer.

Ductal carcinoma in situ (intraductal carcinoma) of the breast is a commonly recognized and curable clinical entity. Patients with intraductal carcinoma are at risk to develop invasive breast cancer presumably due to a transition from the noninvasive to the invasive phase of growth. Primary breast malignancies commonly display both in situ and invasive phases of growth in the same tumor. In the current study, DNA content and alterations in the erbB-2 (HER-2/neu) oncogene product were examined simultaneously in both growth phases of primary breast cancers by image analysis. DNA content in the intraductal and invasive components of primary breast cancers were virtually identical (r = 0.979, p < 0.001). Quantitative image analysis was used to measure erbB-2 expression and categories of expression were related to copy number of the erbB-2 gene. Expression of erbB-2 was similar in both growth phases and implies identity of the erbB-2 genotype. The identity of DNA content suggests that the noninvasive and invasive phases within a single breast cancer are highly related. It is likely that erbB-2 gene number remains the same during progression from intraductal to invasive disease.

Breast Neoplasms↗

PubMed1995

Simultaneous endometrial malignant mixed mesodermal tumor and ovarian serous adenocarcinoma.

Although simultaneous endometrial and ovarian tumors are observed occasionally, rarely are the two neoplasms histologically disparate. We report a case of simultaneous endometrial malignant mixed mesodermal tumor and ovarian serous adenocarcinoma with a single right external iliac lymph node metastasis. Using immunohistochemical profiles, we demonstrated that the two tumors are separate primary neoplasms and identified the origin of the metastatic deposit as the mixed malignant mesodermal tumor.

Cystadenocarcinoma, Serous↗

PubMed1995

Localized adenocarcinoma of the lung: oncogene expression of erbB-2 and p53 in 150 patients.

Historical information and pathological material from 150 consecutive patients with localized adenocarcinoma of the lung was collected to evaluate oncogene expression of erbB-2 and p53, and erbB-2 gene amplification. Pathological material after resection was reviewed to verify histological staging, and patient follow-up was complete in all cases for at least 68 months. Immunohistochemistry of erbB-2 (HER-2/neu) and p53 oncogene expression was performed on two separate paraffin tumor blocks for each patient with normal lung as control. Gene amplification of erbB-2 was measured after DNA extraction from 20-micrometer sections of erbB-2-positive and -negative tumors. All analyses were blinded and included Kaplan-Meier survival estimates with Cox proportional hazards regression modeling. Two adequate blocks of tumor and normal lung were available for 138 (92%) patients. Immunohistochemical identification of expression of p53 was observed in 49 (37%) patients and erbB-2 in 17 (13%) patients. DNA dot blot analyses were performed on 17 erbB-2-positive and 13 randomly selected erbB-2-negative tumors. There was 1 (6%) of 17 erbB-2-positve tumors with 4-fold erbB-2 gene amplification. Actual 5-year survival was 63% and actuarial 10-year survival was 59% for the entire population of 150 patients. Significant univariate predictors (P < 0.05) of cancer death were the presence of symptoms, tumor size >3 cm, poor differentiation, visceral pleural invasion, and p53 expression. Multivariate analysis associated symptoms and p53 expression as independent factors with decreased survival. Thus, this project examined p53 and erbB-2 expression in patients with localized adenocarcinoma and associated p53 status with survival. Multicenter collection of data should allow the development of a model of cancer recurrence in this most common lung cancer.

Adult↗

PubMed1994

Mutation analysis of the THRA1 gene in breast cancer: deletion/fusion of the gene to a novel sequence on 17q in the BT474 cell line.

We have previously described a common region of deletion and allele loss on chromosome 17q in sporadic breast cancers that is likely to contain a tumor suppressor gene. The region, mapped to 17q12-q21, was bordered by D17S250 and D17S579 on the centromeric and telomeric sides, respectively. This deletion region overlaps the BRCA1 locus, which predisposes to familial breast and ovarian cancer. The most frequent loss of heterozygosity was observed at the thyroid hormone receptor alpha (THRA1) locus. Southern analysis revealed a rearrangement of THRA1 in the BT474 breast cancer cell line. This rearrangement represented a deletion of exons 8-10 of one THRA1 allele that was also coamplified with ERBB2. Northern blots showed two mutant transcripts in BT474 cells. Analysis of the mutant transcripts revealed fusion of the THRA1 exon 7 by splicing to a novel sequence designated BTR for "BT474 transcribed rearrangement." BTR was found to be highly conserved and mapped to 17q. The deletion in BT474 cells spans the entire BRCA1 region. To search for additional mutations in the THRA1 gene, all nine protein-encoding exons of THRA1 were examined for point mutations via single strand conformation analysis in a series of primary breast tumors, breast cancer cell lines, and lymphoblastoid cell lines derived from the youngest affected members of several German breast cancer families. No point mutations were detected, including the unrearranged THRA1 allele in BT474. We have thus excluded THRA1 as a commonly mutated sporadic breast cancer tumor suppressor gene and as the BRCA1 gene.

Amino Acid Sequence↗

PubMed1994

Epithelial cells are an important source of tenascin in normal and malignant human breast tissue.

The extracellular matrix glycoprotein tenascin is limited to the periductal matrix of normal breast tissue but is markedly increased in both malignant and fibroadenomatous proliferations. It has been hypothesized that the changes in tenascin expression in these tissues are the result of epithelial induction of tenascin expression by the underlying mesenchyme. We have used Western and Northern blotting techniques to examine tenascin expression by normal and malignant mammary epithelial cells in culture. Normal mammary epithelial cells express tenascin in culture and incorporate the protein into the underlying matrix. The SV40-transformed mammary epithelial cell line HBL100 and some established mammary carcinoma cell lines also express tenascin. In contrast to normal mammary epithelial cells, carcinoma cells incorporate very little tenascin into the underlying matrix. To examine the source of tenascin expression in vivo, we have used in situ hybridization to demonstrate that the epithelial cells are a significant source of tenascin in both normal and malignant breast tissues.

Adenofibroma↗