Early growth response-1 gene: potential radiation response gene marker in prostate cancer.

This study was undertaken to determine whether the transcription factor EGR-1 expression: (1) in the primary tumor, correlates with radiation response in terms of complete local tumor control with no evidence of disease or recurrence and no evidence of metastasis; (2) in the postirradiated biopsies correlates with residual tumor; and (3) correlates with the expression of Egr-1 target genes such as TP53, pRB, and Bax. The authors analyzed: (1) 25 pretreated surgically resected paraffin-embedded primary adenocarcinomas of the prostate for the presence of EGR-1 expression and mutation, and correlated this with clinical endpoints such as serum prostate-specific antigen levels and current clinical status; (2) 27 postirradiated biopsies of prostate for the presence of EGR-1 expression, and correlated these findings to the residual tumor status; and (3) 12 prospective prostate tumor specimens for EGR-1 expression and its target genes. EGR-1 expression was determined by immunohistochemistry and mutations were screened in two regions of the Egr-1 gene (trinucleotide AGC repeats in transactivation domain [TD] and poly A tract in 3'UTR) by polymerase chain reaction-single strand conformational polymorphism analysis. Of 25 patients, 18 patients showed expression of EGR-1. EGR-1 overexpression correlated with treatment failure. No correlation with EGR-1 overexpression and its target genes was found, which may indirectly suggest that overexpressed EGR-1 may lack transactivation function. In summary, EGR-1 overexpression in the mutant form may provide an indication of clinical failure (local recurrence or metastasis).

Reversion of transcriptional repression of Sp1 by 5 aza-2' deoxycytidine restores TGF-beta type II receptor expression in the pancreatic cancer cell line MIA PaCa-2.

The pancreatic cancer cell line, MIA PaCa-2 is not responsive to transforming growth factor beta (TGF-beta) because of a lack of expression of the TGF-beta type II receptor (RII). We show that the lack of RII expression is caused by a deficit of the transcription factor Sp1. Nuclear run-off assays and Western immunoblot showed low levels of transcription and protein levels of Sp1, respectively. Treatment of MIA PaCa-2 cells with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine, resulted in an increase in the rate of Sp1 transcription, in Sp1 protein expression, and in the binding of Sp1 to the RII promoter. Ectopic expression of Sp1 cDNA in MIA PaCa-2 cells led to an increase in RII promoter-chloramphenicol acetyltransferase activity and RII expression. Expression of Sp1 cDNA also caused a reduction in both growth and clonogenicity that was associated with restoration of responsiveness to TGF-beta. Conversely, cells that express RII (BxPC-3 and MIA PaCa-2 Sp1 transfectants) when treated with mithramycin, an inhibitor of Sp1 binding, showed a reduction in RII mRNA expression. The reduction of RII mRNA was attributed to a decrease in RII promoter-chloramphenicol acetyltransferase activity that was associated with a decrease in Sp1 binding to the RII promoter. These data indicate that transcriptional repression of the Sp1 gene in MIA PaCa-2 cells plays a role in the transcriptional inactivation of the RII gene and thus lack of responsiveness to TGF-beta.

Characterization of a newly established human pancreatic carcinoma cell line, UK Pan-1.

BACKGROUND: A highly tumorigenic cell line designated as UK Pan-1 was established in a surgically removed human pancreatic adenocarcinoma and characterized as having many of the genotypic and phenotypic alterations commonly found in pancreatic tumors. METHODS: The cell line was characterized by its morphology, growth rate in monolayer culture and soft agar, tumorigenicity in nude mice, and chromosomal analysis. Furthermore, the status of p53, Ki-ras mutation and transforming growth factor (TGF)-/receptor expression were determined. The characteristics of UK Pan-1 were compared with those of other commonly used pancreatic carcinoma cell lines. RESULTS: Quiescent UK Pan-1 cells could be stimulated to proliferate in growth factor free nutrient media, indicating a growth factor independent phenotype. UK Pan- 1 cells grew in soft agar and rapidly formed tumors in nude mice. This cell line possesses a mutation at codon 12 of the c-Ki-ras-2 gene that is commonly found in pancreatic carcinoma. Fluorescence in situ hybridization showed that two alleles of p53 tumor suppressor gene were present in UK Pan-1. However, sequencing analysis revealed a mutation in one allele at exon 8, codon 273 (G to A; Arg to His). Additional growth assays indicated that the cell line was insensitive to negative growth regulation induced by exogenous TGF-beta. Molecular analysis of the TGF-beta signaling pathway showed that UK Pan-1 did not express appreciable levels of the TGF-beta receptor type I, II, or III mRNAs, but did express DPC4 mRNA. Karyotype analysis revealed an 18q21 deletion indicating a possible loss of heterozygosity for DPC4, as well as other chromosomal deletions and rearrangements. CONCLUSIONS: This study indicates that UK Pan-1 is a highly tumorigenic cell line possessing a molecularly complex pattern of mutations that may be used as a model to further the understanding of the mechanisms responsible for the development of pancreatic carcinoma.

Differential expression of transforming growth factor beta receptors in human pancreatic adenocarcinoma.

BACKGROUND: Many cancer cells show resistance to transforming growth factor beta (TGF-beta)-mediated growth inhibition. This resistance to TGF-beta is associated with an increased tumorigenic phenotype. OBJECTIVE: In this study, we determined whether a loss in expression of TGF-beta receptors or DPC4, an important down stream target of TGF-beta signaling, might account for this lack of TGF-beta sensitivity in pancreatic adenocarcinoma. MATERIALS AND METHODS: To accomplish this, six established pancreatic cancer cell lines, twenty-six surgically resected tumor specimens of pancreatic adenocarcinoma and ten non-tumor pancreas tissues were analyzed for the mRNA expression of the three TGF-beta receptors (RI, RII, and RIII) and DPC4. RESULTS: We report here that five of six pancreatic cancer cell lines were not sensitive to TGF-beta. All the ten non-tumor specimens of pancreas showed expression of RI, and DPC4; while nine of ten showed expression of RIII and eight of ten showed expression of RII. Five of six pancreatic cancer cell lines and 23 of 26 tumor specimens showed expression of RI. Two cell lines and about half (46%) of the tumor specimens did not express RII. Only two cell lines showed appreciable levels of RIII expression; while ten of 26 (38%) tumor specimens did not show expression of RIII. DPC4 expression was observed in three of the six (50%) cell lines and 19 of 24 (79%) tumor specimens. CONCLUSION: This study indicates that apart from the functional loss of DPC4 due to mutations or homozygous deletion, a lack of the TGF-beta receptors, particularly RII and RIII, may contribute to a loss of TGF-beta signaling in a population of pancreatic cancers.

Novel mutations in the polyadenine tract of the transforming growth factor beta type II receptor gene are found in a subpopulation of human pancreatic adenocarcinomas.

In this study, we determined the incidence of microsatellite instability (MIN) in pancreatic adenocarcinoma and determined whether MIN might target, for mutations, the simple nucleotide repeats of the transforming growth factor beta type II receptor (TGFBR2) gene. Forty-eight surgically resected pancreatic tumor tissue samples and two normal pancreas tissue samples were analyzed in this study. Microsatellite analysis was performed for six loci in 14 of the 48 tumor specimens for which we had matching normal genomic DNA. Only four of the 14 tumors (29%) were MIN-positive as determined by the presence of microsatellite variations in more than one locus. Interestingly, eight of the 14 specimens (57%) showed microsatellite variations or loss of heterozygosity at D18S34, suggesting that this locus may be a critical region of genetic instability in pancreatic tumorigenesis. Of the 48 tumors, only two (4%) showed mutations in the polyA region, one of the MIN-targeted sites of the TGFBR2 gene. DNA sequence analysis of these two specimens showed the presence of a two-base deletion in one tumor specimen and the other tumor specimen showed a base substitution in the polyA tract at codon 128 of the TGFBR2 gene. The fact that these mutations occurred in the polyA tract of some pancreatic tumors suggests that a subpopulation of these tumors may be susceptible to MIN-targeted mutations. The incidence of these mutations are low and similar to that reported for nonhereditary, sporadic colon cancers.

Ionizing radiation-inducible apoptosis in the absence of p53 linked to transcription factor EGR-1.

The tumor suppressor protein p53 is a pivotal regulator of apoptosis, and prostate cancer cells that lack p53 protein are moderately resistant to apoptotic death by ionizing radiation. Genes encoding the transcription factor early growth response-1 (EGR-1) and cytokine tumor necrosis factor-alpha (TNF-alpha) were induced upon irradiation of prostate cancer cells, and inhibition of EGR-1 function resulted in abrogation of both TNF-alpha induction and apoptosis. Induction of the TNF-alpha gene by ionizing radiation and EGR-1 was mediated via a GC-rich EGR-1-binding motif in the TNF-alpha promoter. Because TNF-alpha induces apoptosis in prostate cancer cells, these findings suggest that, in the absence of p53, ionizing radiation-inducible apoptosis is mediated by EGR-1 via TNF-alpha transactivation.

EGR-1 induction is required for maximal radiosensitivity in A375-C6 melanoma cells.

Exposure to ionizing radiation leads to induction of the immediate-early gene, early growth response-1 (Egr-1). Previous studies have suggested distinct cell type- and inducer-specific roles for EGR-1 protein in cellular growth inhibition. The present study was undertaken to determine the functional role of EGR-1 in growth inhibition caused by exposure of tumor cells to ionizing radiation. Exposure to ionizing radiation caused induction of EGR-1 protein in human melanoma cells A375-C6. Inhibition of either the function of EGR-1 protein by stable transfection with a dominant-negative mutant or the expression of EGR-1 by transient transfection with an antisense oligomer resulted in a diminished growth-inhibitory response to ionizing radiation. Because previous studies have suggested that mutations in the tumor-suppressor gene p53 confer radio-resistance, we examined the p53 status of A375-C6 cells. Interestingly, both the parental and the transfected A375-C6 cells showed trisomy for wild-type p53 alleles. Exposure to ionizing radiation resulted in induction of p53 protein that localized to the nucleus in A375-C6 cells. These data suggest that inhibition of EGR-1 function confers radio resistance despite the induction of wild-type nuclear p53. Thus, EGR-1 is required for the growth-inhibitory response to ionizing radiation in A375-C6 cells.

C-Ki-ras mutations in peripheral blood of pancreatic cancer patients: a marker for early tumor metastasis.

PURPOSE: To determine the incidence of circulating minimal malignant clone (CMMC) (harboring c-Ki-ras-2 mutation) in peripheral blood (PB) samples of untreated pancreatic cancer using polymerase chain reaction (PCR) analysis of c-Ki-ras-2 oncogene. METHODS AND MATERIALS: Experiments were carried out in fresh tumor, peritoneal washings (PW), and PB samples of untreated pancreatic adenocarcinoma patients (both resectable and unresectable). Samples were taken from 16 patients diagnosed with pancreatic adenocarcinoma for the PCR analysis of mutated c-Ki-ras oncogene. Five tumor samples, 15 PB samples, and 3 PW samples were analyzed for point mutation of the c-Ki-ras gene at codon 12. RESULTS: Out of five tumor samples analyzed for c-Ki-ras mutation, four were positive at the 12th codon. Out of total 15 PB samples, nine were positive for the c-Ki-ras point mutation at the 12th codon. All the positive PB samples showed a base substitution from GGT to GAT in the second position of the 12th codon. Out of three PW samples, two showed mutation at the second position from GGT to GAT similar to their PB and tumor samples. CONCLUSION: Our study indicated the presence of the tumor cells (CMMC) in PB of pancreatic adenocarcinoma that can be identified by PCR analysis of c-Ki-ras oncogene. Patients with presence of CMMC in PB and mutation in PW had aggressive tumors that responded poorly to treatment.