Optimisation of conditions for detection of activated oncogenes by transfection of NIH 3T3 cells.

Optimisation of conditions for detection of activated oncogenes by transfection of NIH 3T3 cells

Ha-ras restriction fragment length polymorphisms in colorectal cancer.

The possibility of an association between restriction fragment length polymorphisms (RFLPs) at the Ha-ras gene locus and susceptibility to develop colorectal cancer has been investigated. Leucocyte DNA from 46 carcinoma patients and 49 controls was analysed by Southern blotting to determine the size distribution of restriction fragments containing the variable tandem repeat 3' to the Ha-ras gene. Four predominant allelic fragments were found in both groups (in AvaII digests having sizes of 1.55, 2.0, 2.65 and 3.15 kilobases [kb]), together with a variety of 'rare' alleles (with individual frequencies less than 5%). The overall prevalence of rare alleles was not significantly different between cancer and control groups. The distribution of the common alleles, however, differed significantly. The combined frequency of the two larger alleles (a3 and a4) was approximately twice as high in the cancer group (34%) as in controls (18%) (P less than 0.025), which was reflected in a highly significant increase in the proportion of individuals carrying an a3 or a4 allele

Cellular senescence in naevi and immortalisation in melanoma: a role for p16?

Cellular senescence, the irreversible proliferative arrest seen in somatic cells after a limited number of divisions, is considered a crucial barrier to cancer, but direct evidence for this in vivo was lacking until recently. The best-known form of human cell senescence is attributed to telomere shortening and a DNA-damage response through p53 and p21. There is also a more rapid form of senescence, dependent on the p16-retinoblastoma pathway. p16 (CDKN2A) is a known melanoma susceptibility gene. Here, we use retrovirally mediated gene transfer to confirm that the normal form of senescence in cultured human melanocytes involves p16, since disruption of the p16/retinoblastoma pathway is required as well as telomerase activation for immortalisation. Expression (immunostaining) patterns of senescence mediators and markers in melanocytic lesions provide strong evidence that cell senescence occurs in benign melanocytic naevi (moles) in vivo and does not involve p53 or p21 upregulation, although p16 is widely expressed. In comparison, dysplastic naevi and early (radial growth-phase, RGP) melanomas show less p16 and some p53 and p21 immunostaining. All RGP melanomas expressed p21, suggesting areas of p53-mediated senescence, while most areas of advanced (vertical growth-phase) melanomas lacked both p16 and p21, implying escape from both forms of senescence (immortalisation). Moreover, nuclear p16 but not p21 expression can be induced in human melanocytes by oncogenic BRAF, as found in around 80% of naevi. We conclude that cell senescence can form a barrier to melanoma development. This also provides a potential explanation of why p16 is a melanoma suppressor gene

Poorly differentiated and anaplastic thyroid carcinomas: chromosomal and oligo-array profile of five new cell lines

Information on gene alterations associated to poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) is scarce. Using human cancer cell lines as a tool for gene discovery, we performed a cytogenetic and oligo-array analysis in five new cell lines derived from two PDTC and three ATC. In PDTC we evidenced, as important, the involvement of the MAPK/ERK kinase pathway, and downregulation of a group of suppressor genes that include E-cadherin. In ATC, downregulation of a specific group of oncosuppressor genes was also observed. Our ATC cell lines presented chromosomal markers of gene amplification, and we were able to identify for the first time the nature of the involved amplicon target genes. We found that the main molecular differences between the two cell line types were related to signal transduction pathways, cell adhesion and motility process. TaqMan experiments performed for five amplicon target genes and for two genes, which allowed a clear distinction between ATC and PDTC: CDH13 and PLAU corroborated array results, not only in the cell lines, but also in an additional set of primary 14 PDTC and three ATC. We suggest that our findings may represent new tools for the development of more effective therapies to the hitherto untreatable ATC

Expression of high p53 levels in colorectal cancer: a favourable prognostic factor

The expression of p53 protein was examined in a series of 111 colorectal cancer adenocarcinomas with a long follow-up. A quantitative luminometric immunoassay (LIA) was used for the measurement of wild-type and mutant p53 protein in extracts from colorectal tumour cytosols, p53 being detected in 42% of the samples (range 0.0–52 ng mg−1). Using an arbitrary cut-off value of 2.7 ng mg−1, 25% of the tumours were classified as manifesting high p53 levels. There was no association of p53 expression with patient age, sex, serum preoperative carcinoembryonic antigen (CEA) levels, tumour site and size, nodal status or TNM stage. Significant and independent correlation was found to exist between high p53 levels and prolonged disease-free survival (P = 0.05) at a median follow-up of 60 months. This survival advantage was most apparent among stage III cancer patients. The results from this study would suggest that expression of high p53 levels appear to be useful in selecting a group of colorectal cancer patients with a better prognosis. © 1999 Cancer Research Campaig

Microsatellite instability in thyroid tumours and tumour-like lesions

Fifty-one thyroid tumours and tumour-like lesions were analysed for instability at ten dinucleotide microsatellite loci and at two coding mononucleotide repeats within the transforming growth factor β (TGF-β) type II receptor (TβRII) and insulin-like growth factor II (IGF-II) receptor (IGFIIR) genes respectively. Microsatellite instability (MI) was detected in 11 out of 51 cases (21.5%), including six (11.7%) with MI at one or two loci and five (9.8%) with Ml at three or more loci (RER+ phenotype). No mutations in the TβRII and IGFIIR repeats were observed. The overall frequency of MI did not significantly vary in relation to age, gender, benign versus malignant status and tumour size. However, widespread MI was significantly more frequent in follicular adenomas and carcinomas than in papillary and Hürthle cell tumours: three out of nine tumours of follicular type (33.3%) resulted in replication error positive (RER+), versus 1 out of 29 papillary carcinomas (3.4%, P = 0.01), and zero out of eight Hürthle cell neoplasms. Regional lymph node metastases were present in five MI-negative primary cancers and resulted in MI-positive in two cases. © 1999 Cancer Research Campaig

Genomic deletion and promoter methylation status of Hypermethylated in Cancer 1 (HIC1) in mantle cell lymphoma

Mantle cell lymphomas (MCL), characterized by the t(11;14)(q13;q32), frequently carry secondary genetic alterations such as deletions in chromosome 17p involving the TP53 locus. Given that the association between TP53-deletions and concurrent mutations of the remaining allele is weak and based on our recent report that the Hypermethylated in Cancer 1 (HIC1) gene, that is located telomeric to the TP53 gene, may be targeted by deletions in 17p in diffuse large B-cell lymphoma (DLBCL), we investigated whether HIC1 inactivations might also occur in MCL. Monoallelic deletions of the TP53 locus were detected in 18 out of 59 MCL (31%), while overexpression of p53 protein occurred in only 8 out of 18 of these MCL (44%). In TP53-deleted MCL, the HIC1 gene locus was co-deleted in 11 out of 18 cases (61%). However, neither TP53 nor HIC1 deletions did affect survival of MCL patients. In most analyzed cases, no hypermethylation of the HIC1 exon 1A promoter was observed (17 out of 20, 85%). However, in MCL cell lines without HIC1-hypermethylation, the mRNA expression levels of HIC1 were nevertheless significantly reduced, when compared to reactive lymph node specimens, pointing to the occurrence of mechanisms other than epigenetic or genetic events for the inactivation of HIC1 in this entity

p21WAF1/CIP1expression in stage I cutaneous malignant melanoma: its relationship with p53, cell proliferation and survival

The expression of p21, p53 and proliferating cell nuclear antigen (PCNA) was analysed by immunohistochemistry in a consecutive series of 369 clinical stage I cutaneous malignant melanoma patients. Correlation of the detected expression levels with each other, with clinicopathological data and with melanoma survival were statistically evaluated. p21 expression was significantly associated with p53 and PCNA expression levels. In addition, high levels of p53 and PCNA were significantly interrelated. Tumour thickness, recurrent disease, high TNM category and older (≥ 55 years) age at diagnosis were inversely associated with p21 expression. Gender, bleeding, tumour thickness, Clark's level of invasion, TNM category and p53 index were all important predictors of both recurrence-free and overall survival of melanoma. In Cox's multivariate analysis including 164 patients with a complete set of data, only high tumour thickness and bleeding predicted poor recurrence-free survival (P= 0.0042 and 0.0087 respectively) or overall survival (P= 0.0147 and 0.0033 respectively). Even though elevated p21 expression may be associated with more favourable prognosis in clinical stage I cutaneous melanoma, our results suggest that cell cycle regulatory effects of p21 can be overcome by some other and stronger, partly yet unknown, mechanisms. 1999 Cancer Research Campaig