Expression of CD1D mRNA transcripts in human choriocarcinoma cell lines and placentally derived trophoblast cells.
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Human placental trophoblast is critically involved in mediating maternal tolerance of the fetal semiallograft. Genes encoding highly polymorphic major histocompatibility complex (MHC) class I and class II antigens that could provoke maternal immune rejection responses are silenced in trophoblast. However, several MHC class I or class I-related products exhibiting reduced or negligible polymorphism are expressed and assumed to be functionally involved in maintaining pregnancy. The CD1 gene family encodes non-polymorphic MHC class I-like products that have the unusual ability to present non-peptide antigens to T cells. One member, CD1D, is expressed in certain epithelial cells and interacts with a specific T-cell subset that may promote the development of Th2-mediated responses believed to be associated with pregnancy. In this study we examined the expression of CD1D in human trophoblast cell lines and placentally derived trophoblast cells by reverse transcriptase-polymerase chain reaction using CD1D-specific oligonucleotide primers. We have found that CD1D mRNA transcripts are expressed in trophoblast cells and cell lines. We have also identified a novel alternatively spliced CD1D mRNA transcript lacking exon 4. Exon 4-intact and exon 4-deficient CD1D transcripts appear to be differentially expressed in different trophoblast and non-trophoblast cell populations. Our studies suggest that at least one member of the CD1 family is transcribed in human trophoblast. (+info)
Induction of the p53-target gene GADD45 in HPV-positive cancer cells.
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The E6 oncoprotein of human papillomaviruses (HPVs) has the potential to functionally antagonize p53. In several experimental model systems, ectopic expression of E6 can block the genotoxic induction of the growth inhibitory p53 target gene gadd45, suggesting that the inactivation of this pathway may play a major role for HPV-associated cell transformation. Here, we investigated whether this reflects the regulation of gadd45 expression in carcinoma-derived HPV-positive cells. We found that the gadd45 gene is efficiently induced by mitomycin C, cisplatin, and UV irradiation in a series of HPV-positive cervical cancer cell lines. Moreover, clear induction of gadd45 gene expression was also observed following treatment with gamma-irradiation, a pathway that is strictly dependent on functional p53. This contrasted with findings in human foreskin keratinocytes experimentally immortalized by expressing the HPV16 E6, E7, or E6/E7 oncogenes from the heterologous CMV promoter, where expression of the E6 gene was linked to a lack of gadd45 induction following gamma-irradiation. These results indicate (1) that the tumorigenic phenotype of HPV-positive cancer cells is not linked to an inability to induce the gadd45 gene following DNA damage, (2) that experimental model systems in which the E6 gene is expressed ectopically and/or in a different cellular context do not necessarily reflect the regulation of p53-associated pathways in HPV-positive cancer cells and (3) that a pathway strictly depending on functional p53 is inducible in HPV-positive cancer cells, providing direct evidence that the endogenous p53 protein in these cells is competent to activate a cellular target gene, despite coexpression of the viral E6 oncogene. (+info)
A novel molecular staging protocol for non-small cell lung cancer.
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A molecular staging protocol using reliable markers is of importance in predicting the prognosis of patients with non-small cell lung cancer (NSCLC) and for instituting their appropriate post-surgical treatment. We analysed tumor tissues from 187 NSCLC patients. The DNA and mRNA were extracted from frozen specimens, and then polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exons 5-8, and mutations of K-ras at exon 1. To determine MRP-1/CD9 gene and KA11/CD82 gene expression, which have been postulated to be metastasis suppressor genes, we have applied quantitative RT-PCR. A Cox multivariate regression analysis showed that nodal status, MRP-1/CD9 and K-ras status were significant factors for prognosis (P<0.0001, P=0.0083 and P=0.0004, respectively). Based on these results, we classified the patients into three groups according to their MRP-1/ CD9 and K-ras status. Patients with both MRP-1/CD9 positive and wild K-ras tumors were defined as group A, patients with either reduced MRP-1/CD9 or mutant K-ras tumors were defined as group B and patients with both reduced MRP-1/CD9 and mutant K-ras tumors were designated as group C. This new classification was significantly correlated with the tumor status and pathological stage (P=0.0098 and P=0.0017, respectively). The overall survival rate of the group A patients was significantly better than the group B patients (59.6% vs 27.9%, P=0.0001) and also that of group B patients was better than the group C patients (27.9% vs 20.0%, P=0.0378). This tendency was also found in patients with 110 node-negative NSCLCs (A vs B vs C=75.8% vs 34.9% vs 0.0%, P<0.0001). A Cox multivariate regression analysis in NSCLC patients demonstrated that an evaluation for both MRP-1/CD9 expression and K-ras mutations had a significant prognostic effect as well as nodal status (P<0.0001). (+info)
Differential involvement of the CD95 (Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells.
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The CD95 (Fas/APO-1) system regulates a number of physiological and pathological processes of cell death. The ligand for CD95 induces apoptosis in sensitive target cells by interacting with a transmembrane cell surface CD95 receptor. We previously reported that the recombinant adenovirus-mediated transfer of the wild-type p53 gene caused apoptotic cell death in a variety of human cancer cells. To better understand the mechanism responsible for this cell death signaling, we have investigated the potential involvement of the CD95 receptor/ligand system in p53-mediated apoptosis. The transient expression of the wild-type p53 gene upregulated the CD95 ligand mRNA as well as protein expression in H1299 human lung cancer cells deficient for p53 and in DLD-1 and SW620 human colon cancer cells with mutated p53, all of which constitutively expressed CD95 receptor as shown by a flow cytometric analysis, and induced rapid apoptotic cell death as early as 24 h after gene transfer. However, the sensitivity to the cytolytic effect of agonistic anti-CD95 antibody (CH11) varied among these cell lines: CH11 induced apoptosis in H1299 cells, but not in DLD-1 and SW620 cells despite their abundant CD95 receptor expression, suggesting that the CD95 receptors on DLD-1 and SW620 cells might be inactivated. In addition, an antagonistic anti-CD95 ligand antibody (4H9) that interfered with the CD95-receptor-ligand interaction partially reduced the apoptosis induced by the wild-type p53 gene transfer in H1299 cells, whereas apoptosis of DLD-1 and SW620 cells occurred in the presence of 4H9. Taken together, these findings led us to conclude that the CD95 receptor/ligand system is differentially involved in p53-mediated apoptosis, suggesting that the restoration of the wild-type p53 function may mediate apoptosis through CD95 receptor/ligand interactions as well as an alternative pathway. (+info)
Truncation of the TGF-beta type II receptor gene results in insensitivity to TGF-beta in human gastric cancer cells.
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The transforming growth factor-beta (TGF-beta receptor system has been implicated in the development of resistance to the growth-inhibitory effects of TGF-beta. It has been reported that resistance to TGF-beta correlates with inactivation of the TGF-beta type II receptor (RII). In the present report, we examine the genetic changes in the TGF-beta RII gene of human gastric cancer cell lines, SNU-5 and SNU-668, which we had previously reported to express truncated TGF-beta RII transcripts. By independent PCR and Southern hybridization analysis of genomic DNA, we found that the genomic sequence of TGF-beta RII is truncated after exon 2 in SNU-5 and after exon 3 in SNU-668. This was confirmed by sequencing the TGF-beta RII cDNA cloned from a SNU-5 cDNA library. Predicted TGF-beta RII protein of SNU-5 cells based on sequencing data contains only a part of extracellular domain of TGF-beta RII. We demonstrate that cotransfection of 3TP-Lux and wild type TGF-beta RII restores the TGF-beta responsiveness in SNU-5 cells, suggesting that genetic changes in the TGF-beta RII gene of SNU-5 cells are responsible for the loss of sensitivity to TGF-beta. This is the first report demonstrating that truncation of the TGF-beta RII gene is an alternative mechanism to inactivate the TGF-beta signal transduction pathways. (+info)
Expression of a new isoform of the tumor susceptibility TSG101 protein lacking a leucine zipper domain in Burkitt lymphoma cell lines.
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The tumor susceptibility gene, TSG101, has been identified as a candidate tumor suppressor gene. We have examined the expression of TSG101 in Burkitt lymphoma cell lines. Several aberrant messages were detected in all cell lines. Aberrant splice donor sites are located within exon 1 at positions 132, 154, 172 and 284. Splice acceptors are located at positions 847 and 1054 within exon 5. The aberrant messages are coexpressed with a normal message and could be the result of additional splicing reactions of the mature message that behaves as an intermediate. The normal message codes for 46 kDa protein (TSG101A). One aberrant message joins in frame nucleotides 283-1055 and codes for a protein isoform of 17 kDa (TSG101B), as demonstrated by in vitro translation assays. The TSG101B isoform lacks the leucine zipper near the C-terminus, a transcriptional repressor domain, and retains most of the N-terminal region which has homology to E2 ubiquitin regulatory enzymes and the CROC-1 transcriptional regulator. The TSG101B isoform was detected in sixteen out of twenty-two (72%) BL cell lines, but not in normal lymphoid populations. The presence of two TSG101 isoforms with different dimerization potential opens up a new level of regulation of the TSG101 proteins possibly affecting cell cycle regulation. (+info)
Correlation between androgen receptor expression and FGF8 mRNA levels in patients with prostate cancer and benign prostatic hypertrophy.
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AIM: To investigate the correlation between androgen receptor expression and fibroblast growth factor 8 (FGF8) mRNA levels. METHODS: 39 human prostate cancers and 14 benign prostatic hypertrophy specimens were examined immunohistochemically for androgen receptor expression and by in situ hybridisation and reverse transcription polymerase chain reaction for FGF8 expression. RESULTS: In 39 tumours there was a statistically significant negative correlation between tumour grade and FGF8 expression and a positive correlation between FGF8 and androgen receptor expression. All 14 benign hypertrophy specimens expressed moderate to high levels of FGF8 and androgen receptor. CONCLUSIONS: Loss of FGF8 may be a factor involved in the development of prostatic cancer. (+info)
Overexpression and amplification of c-myc in the Syrian hamster kidney during estrogen carcinogenesis: a probable critical role in neoplastic transformation.
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An estrogen receptor-driven, multistep process for estrogen carcinogenesis in the Syrian hamster kidney is proposed. Because in this species the reproductive and urogenital tracts arise from the same embryonic germinal ridge, it is evident that the kidney has carried over genes that are responsive to estrogens. Using in situ hybridization, overexpression of early estrogen-response genes, i.e., c-myc and c-fos, has been shown to be localized preferentially in early renal tumor foci after 3.5-4.0 months of estrogen treatment. This event coincides with an increased number of S-phase proliferating cell nuclear antigen-labeled cells in these tumor foci, along with a rapid rise in aneuploid frequency in the kidney. Western blot analyses of c-MYC and c-FOS protein products support the overexpression of these genes. Amplification of c-myc, 2.4-3.6-fold, but not of c-fos, was detected in 67% of the primary renal tumors examined, by Southern blot analyses. Consistent chromosomal gains, common to both diethylstilbestrol- and estradiol-induced renal neoplasms, were observed in chromosomes 1, 2, 3, (6), 11, (13), 16, 20, and 21 (chromosome number alterations are indicated in parentheses). Using fluorescence in situ hybridization, the c-myc gene was localized to hamster chromosome 6qb. Chromosome 6 exhibited a high frequency of trisomies and tetrasomies in the kidney after 5.0 months of estrogen treatment and in primary renal tumors. The data presented indicate that estrogen-induced genomic instability may be a key element in carcinogenic processes induced by estrogens. (+info)