Andrey Frolov

Publications of Andrey Frolov

• ErbB3 expression promotes tumorigenesis in pancreatic adenocarcinoma.

  Authors: J Spencer Liles, Juan Pablo Arnoletti, Ching-Wei D Tzeng, J Harrison Howard, Andrew V Kossenkov, Peter Kulesza, Martin J Heslin, Andrey Frolov

  Cancer biology & therapy. 09/2010; 10(6):555-63.

  Historically, ErbB3 has been overlooked within the ErbB receptor family due to its perceived lack of tyrosine kinase activity. We have previously demonstrated that in pancreatic cancer ErbB3 is theHistorically, ErbB3 has been overlooked within the ErbB receptor family due to its perceived lack of tyrosine kinase activity. We have previously demonstrated that in pancreatic cancer ErbB3 is the preferred dimerization partner of EGFR, ErbB3 protein expression level directly correlates with the anti-proliferative effect of erlotinib (an EGFR-specific tyrosine kinase inhibitor), and transient knockdown of ErbB3 expression results in acquired resistance to EGFR-targeted therapy. In this study, we develop a stable isogenic model of ErbB3 expression in an attempt to decipher ErbB3's true contribution to pancreatic cancer tumorigenesis and to examine how this receptor affects cellular sensitivity to EGFR-targeted therapy. Analysis of the EGFR-ErbB3 heterodimer demonstrates that ligand-induced PI3K-AKT signaling is limited to ErbB3-expressing cells and that this signaling cascade can be partially abrogated by inhibiting EGFR function with erlotinib. Using our model of exogenous ErbB3 expression we showed a direct relationship between ErbB3 protein levels and increased pancreatic cancer cell proliferation in vitro. In vivo, ErbB3(+)PANC-1 xenografts had a significantly larger tumor volume than PANC-1 control xenografts (ErbB3-PANC-1) and displayed increased sensitivity to EGFR-targeted therapy. In pancreatic cancer, ErbB3 appears to be critically involved in EGFR signaling as evidenced by its profound effect on cellular proliferation and its ability to influence response to EGFR-targeted therapy.

• Epidermal growth factor receptor (EGFR) intron 1 polymorphism and clinical outcome in pancreatic adenocarcinoma.

  Authors: Andrey Frolov, J Spencer Liles, Andrew V Kossenkov, Ching-Wei D Tzeng, Nirag Jhala, Peter Kulesza, Shyam Varadarajulu, Mohamad Eloubeidi, Martin J Heslin, J Pablo Arnoletti

  American journal of surgery. 09/2010; 200(3):398-405.

  Epidermal growth factor receptor (EGFR) intron 1 has a polymorphic region of CA repeats that is believed to be associated with increased EGFR expression, tumor aggressiveness, and worse survival inEpidermal growth factor receptor (EGFR) intron 1 has a polymorphic region of CA repeats that is believed to be associated with increased EGFR expression, tumor aggressiveness, and worse survival in cancer patients. A large population of pancreatic adenocarcinoma patients was investigated to evaluate this polymorphism as a potential prognostic marker of clinical outcome. Deoxyribonucleic acid obtained from 50 resected pancreatic adenocarcinomas and from 85 diagnostic endoscopic ultrasound-guided fine-needle aspiration procedures corresponding to patients with unresectable tumors was included. The correlation between CA repeat length and EGFR messenger ribonucleic acid levels was also examined. Analysis of the 135 patients revealed no correlation between EGFR intron 1 CA repeat length and tumor stage. There was no difference in overall patient survival when stratified by allele length. A correlation between EGFR intron 1 length and EGFR transcript and protein levels could not be established. The length of the EGFR intron 1 CA repeats does not correlate with levels of EGFR expression and cannot be used as marker of clinical prognosis in pancreatic cancer patients.

• Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells.

  Authors: Callinice D Capo-Chichi, Jennifer L Smedberg, Malgorzata Rula, Emmanuelle Nicolas, Anthony T Yeung, Richard F Adamo, Andrey Frolov, Andrew K Godwin, Xiang-Xi Xu

  Stem cells international. 01/2010; 2010:602068.

  Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonicBackground. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment. Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (-/-) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (-/-) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (-/-) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages. Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.

• Epigenetic downregulation of the DNA repair gene MED1/MBD4 in colorectal and ovarian cancer.

  Authors: J Harrison Howard, Andrey Frolov, Ching-Wei Tzeng, Ashley Stewart, Andrew Midzak, Amar Majmundar, Andrew Godwin, Martin Heslin, Alfonso Bellacosa, J Pablo Arnoletti

  Cancer biology & therapy. 01/2009; 8(1).

  MED1 is a base excision repair enzyme that interacts with the mismatch repair protein MLH1 and maintains genomic integrity by binding methylated DNA and repairing spontaneous deamination events. MED1MED1 is a base excision repair enzyme that interacts with the mismatch repair protein MLH1 and maintains genomic integrity by binding methylated DNA and repairing spontaneous deamination events. MED1 mutations have been associated with microsatellite instability and accelerated colorectal cancer (CRC) tumorigenesis. We propose that promoter methylation may serve as an alternative epigenetic mechanism for MED1 gene suppression during sporadic CRC tumorigenesis. Methylation status of the MED1 promoter was investigated in a panel of ovarian and colorectal cancer cell lines. The MED1 promoter region was sequenced following bisulfite treatment and sequence analysis identified a CpG island within the MED1 promoter which is frequently and preferentially methylated (>/=50%) in ovarian and colorectal cancer cell lines with low/reduced MED1 expression. In vitro reversal of methylation restored MED1 expression. In colorectal cancer patients, when MED1 methylation was present, both tumor and matched mucosa were affected equally (mean frequency of methylation 24%) and there was no correlation between methylation and tumor stage. Patients without history of CRC showed significantly lower frequency of methylation (mean 14%, p < 0.05). Decreased MED1 transcript levels were observed in matched normal mucosa when compared to controls (median fold difference 8.0). Additional decreased expression was seen between mucosa and matched tumor (median fold decrease 4.4). Thus, MED1 promoter methylation and gene silencing occur in sporadic CRC patients and represent an early event in CRC tumorigenesis. Detection of MED1 methylation and gene suppression in normal colon mucosa may contribute to identifying patients at higher risk of developing CRC during screening procedures.

• Molecular Mechanisms of Action of Imatinib Mesylate in Human Ovarian Cancer: A Proteomic Analysis.

  Authors: Bhavinkumar Patel, Yin He, Xin-Ming Li, Andrey Frolov, Lisa Vanderveer, Carolyn Slater, Russel Schilder, Margaret von Mehren, Andrew Godwin, Anthony Yeung

  Cancer genomics & proteomics. 08/2008; 5(3-4):137-150.

  Background: Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland) is a small-molecule tyrosine kinase inhibitor with activity against ABL, BCR-ABL, c-KIT, and PDGFRalpha. Several clinical trialsBackground: Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland) is a small-molecule tyrosine kinase inhibitor with activity against ABL, BCR-ABL, c-KIT, and PDGFRalpha. Several clinical trials have evaluated the efficacy and safety of imatinib in patients with ovarian carcinoma who have persistent or recurrent disease following front-line platinum/taxane based chemotherapy. However, there is limited pre-clinical and clinical data on the molecular targets and action of imatinib in ovarian cancer. Materials and Methods: Human ovarian cancer cells (A2780) were treated with imatinib mesylate for either 6 or 24 h. We employed a 2D (two-dimensional) gel electrophoresis and mass spectrometry-based proteomics approach to identify protein expression patterns and signaling pathways that were altered in response to imatinib. Cells were analyzed for PDGFRalpha; and AKT expression, which were then correlated with imatinib sensitivity. Results: Using 2D gel electrophoresis of overlapping pH ranges from pH 4 to 11, about 4,000 protein spots could be analyzed reproducibly. Proteins whose levels changed between two fold to 30 fold were grouped according to whether changes were in the same direction at both time points of treatment with respect to the control, or changed their levels only at one of the time points. Conclusion: Differentially regulated proteins following imatinib treatment of A2780 cells involved the regulation of actin cytoskeleton, metabolic pathways, cell cycle, cell proliferation, apoptosis, cell junctions, and signal transduction. Thus, exposure of cells to imatinib produces complex changes in the cell that require further investigation.

• EGFR genomic gain and aberrant pathway signaling in pancreatic cancer patients.

  Authors: Ching-Wei D Tzeng, Andrey Frolov, Natalya Frolova, Nirag C Jhala, J Harrison Howard, Selwyn M Vickers, Donald J Buchsbaum, Martin J Heslin, J Pablo Arnoletti

  The Journal of surgical research. 12/2007; 143(1):20-6.

  BACKGROUND: Epidermal growth factor receptor (EGFR) expression does not predict response to anti-EGFR therapy with erlotinib in pancreatic cancer patients. In the absence of known pancreatic cancerBACKGROUND: Epidermal growth factor receptor (EGFR) expression does not predict response to anti-EGFR therapy with erlotinib in pancreatic cancer patients. In the absence of known pancreatic cancer EGFR activating mutations, we sought to identify alternative factors, such as increased gene copy number (genomic gain) and ligand overexpression, which could be associated with aberrant EGFR pathway activation and improved response to targeted therapy. METHODS: EGFR gene copy number was analyzed by fluorescence in situ hybridization in nine pancreatic cancer cell lines and 31 pancreatic cancer surgical specimens. In vitro effects of erlotinib on tumor cell proliferation were tested. Tumor specimen EGFR expression levels were measured by reverse transcriptase-polymerase chain reaction. Expression of stimulating ligand (EGF), phosphorylated receptor (p-EGFR), and activated downstream adaptor proteins (p-Akt and p-ERK), were evaluated by immunohistochemistry and immunoblotting. RESULTS: Pancreatic cancer EGFR genomic gain, in the form of high polysomy, was present in four of nine cell lines and in 10/24 (42%) of patients. Twenty-four patients (77%) expressed EGFR transcript, and of those, half displayed p-EGFR (35% of all patients). A majority of patients demonstrated downstream EGFR pathway activation, with 65% expressing p-Akt and 84% expressing p-ERK. EGFR-expressing tumors also expressed EGF, with exclusive tumor cell localization, suggesting autocrine stimulation of the EGFR pathway. EGF expression level was significantly greater in patients with increased EGFR gene copy number (P = 0.016). CONCLUSIONS: Increased EGFR gene copy number and elevated EGF levels are present in a significant proportion of pancreatic cancer patients, and this may reflect increased EGFR pathway dependence with improved sensitivity to EGFR-targeted therapy.

• Pancreatic cancer epidermal growth factor receptor (EGFR) intron 1 polymorphism influences postoperative patient survival and in vitro erlotinib response.

  Authors: Ching-Wei D Tzeng, Andrey Frolov, Natalya Frolova, Nirag C Jhala, J Harrison Howard, Selwyn M Vickers, Donald J Buchsbaum, Martin J Heslin, J Pablo Arnoletti

  Annals of surgical oncology. 07/2007; 14(7):2150-8.

  BACKGROUND: Epidermal growth factor receptor (EGFR) has a highly polymorphic CA repeat region that affects transcription efficiency and anti-EGFR drug sensitivity in carcinomas. Erlotinib is an EGFRBACKGROUND: Epidermal growth factor receptor (EGFR) has a highly polymorphic CA repeat region that affects transcription efficiency and anti-EGFR drug sensitivity in carcinomas. Erlotinib is an EGFR tyrosine kinase inhibitor approved for pancreatic cancer treatment. We analyzed the impact of EGFR intron 1 CA repeat lengths in pancreatic cancer clinical outcome and in vitro response to erlotinib. METHODS: Allele-specific EGFR intron 1 lengths were analyzed in 30 microdissected pancreatic cancer surgical specimens, matched peripheral blood samples, and 9 pancreatic cancer cell lines treated with erlotinib. CA repeat lengths were correlated with survival, tumor parameters, molecular markers of EGFR pathway activation, and in vitro antiproliferative effects of erlotinib. RESULTS: Both patient samples and cell lines displayed the full spectrum of EGFR CA repeat lengths (14-22 per allele). Patients with shorter sum of total CA repeats (<36) had worse median survival than patients with >or=36 repeats (13.7 vs 30.6 months, P = .002). Shorter patient EGFR intron 1 length correlated with EGFR expression (P = .026). Tumor intron 1 length was identical to that of matched peripheral blood specimens. There was no correlation between EGFR intron 1 length and pancreatic cancer stage, nodal status, grade, or expression of p-EGFR, p-ERK and p-Akt. Shorter EGFR intron 1 length was associated with in vitro response to erlotinib treatment (P = .02). CONCLUSIONS: Shorter EGFR intron 1 CA repeat length is associated with worse pancreatic cancer clinical prognosis and in vitro response to erlotinib. EGFR intron 1 length can be reliably measured in peripheral blood and may translate into a quantitative predictive marker of both pancreatic cancer aggressiveness and erlotinib sensitivity.

• Epidermal growth factor receptor (EGFR) is highly conserved in pancreatic cancer.

  Authors: Ching-Wei D Tzeng, Andrey Frolov, Natalya Frolova, Nirag C Jhala, J Harrison Howard, Donald J Buchsbaum, Selwyn M Vickers, Martin J Heslin, J Pablo Arnoletti

  Surgery. 05/2007; 141(4):464-9.

  BACKGROUND: Pancreatic cancer remains a deadly disease, and the vast majority of pancreatic cancer patients are not candidates for treatment with curative intent. Erlotinib, an EGFR-specific tyrosineBACKGROUND: Pancreatic cancer remains a deadly disease, and the vast majority of pancreatic cancer patients are not candidates for treatment with curative intent. Erlotinib, an EGFR-specific tyrosine kinase inhibitor, was approved recently for use in patients with pancreatic cancer. Somatic mutations in the EGFR gene appear to predict survival and response to tyrosine kinase inhibitor therapy in a subset of patients with non-small-cell lung cancer (NSCLC). METHODS: The purpose of this study was to characterize EGFR mutations in pancreatic adenocarcinoma. EGFR TK mutations were analyzed in 9 pancreatic carcinoma cell lines and 31 clinical specimens from patients with pancreatic cancer who underwent resection. Using laser capture microdissection, tumor cells from patients were harvested selectively for genomic DNA extraction. Mutations were examined by direct sequencing of exons 18-21. RESULTS: Of 9 pancreatic cancer cell lines, 6 had either 2454G>A or 2361G>A mutations in exon 20. Of 31 patients, 25 patients had 2361G>A in exon 20, and 1 patient had 2508C>T in exon 21. All were silent mutations. CONCLUSIONS: The EGFR tyrosine kinase domain is highly conserved in pancreatic cancer. The association among EGFR mutation status, clinical prognosis, and response to anti-EGFR therapy described in NSCLC may not be applicable to pancreatic cancer. This observation does not diminish the possible role of anti-EGFR therapy in pancreatic cancer because there remains a need to explore alternative explanations for pancreatic cancer aberrant EGFR pathway activation such as ligand overexpression, gene amplification, and loss of inhibitory mechanisms.

• ErbB3 expression and dimerization with EGFR influence pancreatic cancer cell sensitivity to erlotinib.

  Authors: Andrey Frolov, Kyle Schuller, Ching-Wei D Tzeng, Emily E Cannon, Brandon C Ku, J Harrison Howard, Selwyn M Vickers, Martin J Heslin, Donald J Buchsbaum, J Pablo Arnoletti

  Cancer biology & therapy. 05/2007; 6(4):548-54.

  Abnormal expression and signaling of ErbB receptors has been implicated in multiple epithelial malignancies, including pancreatic cancer. Erlotinib, an epidermal growth factor receptor tyrosineAbnormal expression and signaling of ErbB receptors has been implicated in multiple epithelial malignancies, including pancreatic cancer. Erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has been recently approved for pancreatic cancer treatment, but there are no reliable predictors of patient response. Expression of additional ErbB receptors seems to influence tumor response to EGFR-targeted therapy. We analyzed the influence of ErbB3 expression on pancreatic cancer cell response to erlotinib treatment. Proliferation assays of five human pancreatic cancer cell lines were performed following treatment with erlotinib. Expression and phosphorylation profiles of ErbB receptors and downstream adaptor protein (Akt, ERK1/2, STAT3, mTOR) were evaluated following stimulation with EGF or neuregulin-beta. The formation of EGFR homodimers and EGFR-ErbB3 heterodimers, necessary to enable ErbB3 downstream signaling, was demonstrated by chemical cross-linking assays. The effects of RNA inhibition of ErbB3 on sensitivity to erlotinib treatment were evaluated in AsPC-1 pancreatic cancer cells. Erlotinib inhibited Akt phosphorylation and proliferation of all the ErbB3-expressing cell lines but did not affect mTOR activation. Cross-linking studies confirmed the presence of EGFR-ErbB3 heterodimers in pancreatic cancer cells. Only the ErbB3-deficient MIA PaCa-2 cells displayed persistent Akt activation and ongoing proliferation in spite of erlotinib treatment. siRNA-mediated inhibition of ErbB3 expression in AsPC-1 cells resulted in acquired resistance to erlotinib treatment. Pancreatic cancer cells which lack ErbB3 do not display activation of the ErbB3-PI3K-Akt cascade induced by EGFR/ErbB3 heterodimers and become less critically dependent on EGFR signaling and therefore resistant to erlotinib. Pancreatic cancer expression of ErbB3 may be useful for EGFR-targeted therapy patient selection.

• Tumor-associated down-regulation of 15-lipoxygenase-1 is reversed by celecoxib in colorectal cancer.

  Authors: Martin J Heslin, Ashley Hawkins, William Boedefeld, J Pablo Arnoletti, Andrey Frolov, Richie Soong, Marshall M Urist, Kirby I Bland

  Annals of surgery. 06/2005; 241(6):941-6; discussion 946-7.

  OBJECTIVE: To evaluate the role of celecoxib on 15-lipoxygenase-1 (15-LOX-1) expression, protein levels, and rates of apoptosis in colorectal cancer cell lines. Also, to evaluate the expression ofOBJECTIVE: To evaluate the role of celecoxib on 15-lipoxygenase-1 (15-LOX-1) expression, protein levels, and rates of apoptosis in colorectal cancer cell lines. Also, to evaluate the expression of 15-LOX-1 in human normal mucosa, adenoma, and carcinoma with correlation to overall survival. SUMMARY BACKGROUND DATA: The function of 15-LOX-1 is to maintain normal rates of apoptosis (programmed cell death). Decreased apoptosis is one mechanism of cancer growth and dissemination. It is our hypothesis that expression of 15-LOX-1 is reduced in human colorectal cancer (CRC) and the administration of celecoxib can reverse this process and induce apoptosis. METHODS: Effect of celecoxib in cell culture: The effect of 40 micromol/L celecoxib was compared with untreated controls in tissue culture utilizing HT-29 and DLD-1 CRC cell lines. Expression of 15-LOX-1 protein was measured by immunoblot. Induction of apoptosis was evaluated by annexin V staining. All data are presented as mean +/- SEM, with significance defined as P < 0.05. 15-LOX-1 in human CRC: From February 1998 to January 2002, 126 patients underwent surgical resection of either colorectal adenomas (n = 24) or carcinomas (n = 102), or both (n = 25). Tissue was macrodissected, snap frozen, and stored at -80 degrees C. After tissue processing, RNA was extracted and gene expression of 15-LOX-1 was quantified utilizing ABI prism real-time quantitative RT-PCR. Significance evaluated by the Wilcoxon signed rank test. RESULTS: Effect of celecoxib in cell culture: After 72 hours of treatment with celecoxib, immunoblot demonstrated a 1.5- to 2-fold increase in 15-LOX-1 protein expression in HT-29 and DLD-1 cells, respectively. Celecoxib produced greater than a 2-fold increase in the rate of apoptosis compared with control cells in both cell lines (P < 0.05). 15-LOX-1 in human CRC: The mean age of the patients was 62 +/- 1 years; 78% were white and 48% were female. The mean size of the polyps and cancers were 3.0 +/- 0.4 and 5.0 +/- 0.1 cm, respectively. Expression of 15-LOX-1 relative to S9 was 30 in normal mucosa and significantly down-regulated to 11 in adenomas and 16 in carcinomas (P < 0.05). CONCLUSIONS: 15-LOX-1 gene expression is significantly reduced in both human colorectal adenomas and carcinomas and associated with decreased survival. Administration of celecoxib restores 15-LOX-1 protein expression and induces apoptosis. Down-regulation of 15-LOX-1 is an early event in the adenoma to carcinoma sequence, and reversal with celecoxib may represent one mechanism for chemoprevention of polyps or treatment of carcinomas.

• Response markers and the molecular mechanisms of action of Gleevec in gastrointestinal stromal tumors.

  Authors: Andrey Frolov, Santiago Chahwan, Michael Ochs, Juan Pablo Arnoletti, Zhong-Zong Pan, Olga Favorova, Jonathan Fletcher, Margaret von Mehren, Burton Eisenberg, Andrew K Godwin

  Molecular cancer therapeutics. 09/2003; 2(8):699-709.

  Gastrointestinal stromal tumors (GISTs), defined by the presence of constitutively activated KIT, are the most common gastrointestinal mesenchymal malignancies. This observation has been successfullyGastrointestinal stromal tumors (GISTs), defined by the presence of constitutively activated KIT, are the most common gastrointestinal mesenchymal malignancies. This observation has been successfully exploited in clinical trials of Gleevec (also known as imatinib mesylate, STI-571) for patients with unresectable and/or metastatic GISTs. The biological mechanisms of Gleevec as well as its downstream molecular effects are generally unknown. We used a DNA microarray-based approach to identify gene expression patterns and signaling pathways that were altered in response to Gleevec in GIST cells. We identified a total of 148 genes or expressed sequence tags (of 10,367) that were differentially regulated; 7 known genes displayed a durable response after treatment. The significantly down-regulated genes were SPRY4A, FZD8, PDE2A, RTP801, FLJ20898, and ARHGEF2. The only up-regulated gene was MAFbx. On a functional level, we demonstrated that imatinib inhibited phosphorylation of KIT, AKT, and extracellular signal-regulated kinase 1/2 without affecting the total level of these proteins and that differential expression of these response genes involved activation of mitogen-activated protein kinase-dependent and -independent pathways. In an attempt to correlate these in vitro findings to clinical data, we examined GIST needle biopsy specimens taken from patients before and after Gleevec administration according to the CSTI571-B2222 Phase II trial and demonstrated that expression levels of the two gene transcripts evaluated correlated well with clinical response. This study emphasizes the potential value of an in vitro cell model to investigate GIST response to imatinib in vivo, for the purpose of identifying important genetic markers of clinical response, mechanisms of drug action, and possible therapeutic targets.

• DNA array-based method for detection of large rearrangements in the BRCA1 gene.

  Authors: Andrey Frolov, Amanda H Prowse, Lisa Vanderveer, Betsy Bove, Hong Wu, Andrew K Godwin

  Genes, chromosomes & cancer. 12/2002; 35(3):232-41.

  In most families with multiple cases of breast and ovarian cancer, the cancer appears to be associated with germline alterations in BRCA1 or BRCA2. However, somatic mutations in BRCA1 and BRCA2 inIn most families with multiple cases of breast and ovarian cancer, the cancer appears to be associated with germline alterations in BRCA1 or BRCA2. However, somatic mutations in BRCA1 and BRCA2 in sporadic breast and ovarian tumors are rare, even though loss of heterozygosity in BRCA1 and BRCA2 loci in these tumors appears frequently. This may be attributed to mutation detection assays that detect alterations in the coding regions and splice site junctions, but that miss large gene rearrangements. To look specifically for mutations such as large gene rearrangements that span several kilobases (kb) of genomic DNA, we have developed a fluorescence DNA microarray assay. This assay rapidly and simultaneously screens for such rearrangements along the entire gene. In our screen of 15 malignant ovarian tumors, we found one sample with a novel 3-kb deletion encompassing exon 17 of BRCA1 that leads to a frameshift mutation. This deletion was not detected in the corresponding constitutive DNA. Our results indicate that, whereas somatic mutations in BRCA1 appear to be rare in ovarian cancers, the search for large gene rearrangements should be included in any BRCA1 mutational analysis. Furthermore, the method described in this report has the potential to screen clinical tumor samples for genomic rearrangements simultaneously in a large number of cancer-associated genes.

• Ras/MAPK pathway confers basement membrane dependence upon endoderm differentiation of embryonic carcinoma cells.

  Authors: Jennifer L Smedberg, Elizabeth R Smith, Callinice D Capo-Chichi, Andrey Frolov, Dong-Hua Yang, Andrew K Godwin, Xiang-Xi Xu

  The Journal of biological chemistry. 11/2002; 277(43):40911-8.

  The formation of extraembryonic endoderm is one of the earliest steps in the differentiation of pluripotent cells of the inner cell mass during the early stages of embryonic development. TheThe formation of extraembryonic endoderm is one of the earliest steps in the differentiation of pluripotent cells of the inner cell mass during the early stages of embryonic development. The primitive endoderm cells and the derived parietal and visceral endoderm cells gain the capacity to produce collagen IV and laminin. The deposition of these components results in the formation of basement membrane and epithelium of the endoderm, with polarized cells covering the inner surface of the blastocoels. We used retinoic acid-induced endoderm differentiation of stem cell-like F9 embryonic carcinoma cells to study the role of the Ras pathway and its regulation in the formation of the visceral endoderm. Upon endoderm differentiation of F9 cells induced by retinoic acid, c-Fos expression, the downstream target of the Ras pathway, is suppressed by uncoupling Elk-1 phosphorylation/activation to MAPK activity. However, attachment to matrix gel greatly enhances the activation of MAPK in endoderm cells but not in undifferentiated F9 cells. Enhanced MAPK activation as a result of contact with basement membrane is able to compensate for reduced Elk-1 phosphorylation and c-Fos expression. We conclude that endoderm differentiation renders the activation of the Ras pathway basement membrane dependent, contributing to the epithelial organization of the visceral endoderm.