We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin Dl gene. We also examined 36 of these 50 carcinomas for expression of the cydin Dl and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3-to 10-fold amplification of the cyclin Dl INT2,, and loss of heterozygosity of the tumor suppressor genes RB (9), p53 (10-12), MCC (13) and APC (13). Point mutations in the p53 gene occur in about 40% of esophageal cancers (10-12). However, activation of RAS oncogenes by point mutation appears to be a very rare event in this type of cancer (12,(14)(15)(16). Despite the fact that amplification of the HSTI and INT2 genes on chromosome 11q13 has been found in about 20-50% ofesophageal cancers, little or no expression of these two genes has been detected in the corresponding cells (6)(7)(8). These findings suggest that an additional gene(s) at the chromosome 11q13 locus is involved in the development of esophageal cancer. These considerations attracted our interest in a recent report thatThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. the cyclin Dl gene had been mapped to the chromosome 11q13 locus close to the INT2 and HSTI genes (17).Cyclins form a family of proteins that complex with cyclindependent protein kinases (CDKs) to govern key transitions in the cell cycle (for review, see refs. 18 and 19) Cyclin Dl was isolated as a gene that is rearranged in parathyroid adenomas (PRADI) and certain B-cell leukemias (BCL-J) (17,20). It also complements a Saccharomyces cerevisiae strain that is mutant in three known Gl cyclins (21, 22) and is induced in the late G1 phase following the treatment of a growth-arrested macrophage cell line with colony-stimulating factor 1 (23). The product of the cyclin Dl gene is expressed at high levels and forms a complex with a CDK during the G1 phase of the cell cycle in sychronized cells (23). In a recent study we detected amplification of the cyclin Dl gene in about 25% of primary esophageal tumors from China (24).The RB gene, which is located on chromosome 13q14, was the first tumor suppressor gene to be isolated. Loss of heterozygosity and loss ofexpression ofthe RB gene are seen during the development of retinoblastoma (RB) and several other types of human cancers (25). The product of the RB gene is a 110-kDa nuclear phosphoprotein. This protein is hypophosphorylated during the G1 phase and hyperphosphorylated in the S, G2, and M phases of the cell cycle. It is thought that this phosphorylation blocks an inhibitory function ofthe RB protein on progression through the later phases of the cell cycle (26). Alternatively, oncoproteins encoded by certain DNA tumor viruses can bind to the RB protein and block its inhibitory function (26). In vitro studies demonstrate that several serine and th...

In recent years, there has been an exponential increase in the number of p53 mutations identified in human cancers. The p53 mutation database consists of a list of point mutations in thep53 gene of human tumors and cell lines, compiled from the published literature and made available through electronic media. The database is now maintained at the International Agency for Research on Cancer (IARC) and is updated twice a year. The current version contains records on 5091 published mutations and is expected to surpass the 6000 mark in the January 1997 release. The database is available in various formats through the European Bioinformatics Institute (EBI) ftp server at: ftp://ftp.ebi.ac.uk/pub/databases/p53/ or by request from IARC (p53database@iarc.fr) and will be searchable through the SRS system in the near future. This report provides a description of the criteria for inclusion of data and of the current formats, a summary of the relevance ofp53 mutation analysis to clinical and biological questions, and a brief discussion of the prospects for future developments.

Summary Vascular endothelial growth factor (VEGF) expression and mutations of cancer-related genes increase with cancer progression. This correlation suggests the hypothesis that oncogenes and tumour suppressors regulate VEGF, and a significant correlation between p53 alteration and increased VEGF expression in human lung cancer was reported recently. To further examine this hypothesis, we analysed VEGF protein expression and mutations in p53 and K-ras in 27 non-small-cell lung cancers (NSCLC): 16 squamous cell, six adenocarcinomas, one large cell, two carcinoids and two undifferentiated tumours. VEGF was expressed in 50% of the squamous cell carcinomas (SCC) and carcinoids but none of the others. p53 mutations occurred in 14 tumours (52%), and K-ras mutations were found in two adenocarcinomas and one SCC; there was no correlation between the mutations and VEGF expression. As nitric oxide also regulates angiogenesis, we examined NOS expression in NSCLC. The Ca2+-dependent NOS activity, which indicates NOS1 and NOS3 expression, was significantly reduced in lung carcinomas compared with adjacent non-tumour tissue (P < 0.004). Although the Ca2+-independent NOS activity, which indicates NOS2 expression, was low or undetectable in non-tumour tissues and most carcinomas, significant activity occurred in three SCC. In summary, our data do not show a direct regulation of VEGF by p53 in NSCLC. Finally, we did not find the up-regulation of NOS isoforms during NSCLC progression that has been suggested for gynaecological and breast cancers.

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Cycin D1 has been implicated in G cell cycle Since the major regulatory events leading to mammalian cell proliferation and differentiation occur in the Go-to-Gi and/or at the G1-to-S phase transition of the cell cycle (1), the deregulated expression of G1 or G1/S phase cyclins or their related cyclin-dependent kinases (CDKs) might cause loss of cell cycle control and thus enhance carcinogenesis. The strongest connection between cyclins and carcinogenesis comes from studies on cyclin D1. The cyclin D1 gene was isolated as a gene that is rearranged and overexpressed in parathyroid adenomas (2, 3). In independent studies, cyclin D1 also rescued a G1 cyclin-defective Saccharomyces cerevisiae strain, and its expression was induced in G1 by growth factors (4, 5). Microinjection and electroporation of anti-cyclin D1 antibodies into mammalian cells revealed that cyclin D1 is essential for cell cycle progression in G1 (5, 6). Amplification and overexpression of cyclin D1 were detected in several human carcinomas, including esophageal carcinomas (6).Transforming growth factor 8s (TGF-ps) are prototypic multifunctional negative growth factors that inhibit epithelial cell proliferation by delaying or arresting progression through the late portion of G1 (7). It is believed that one of the mechanisms whereby cells undergo neoplastic transformation and escape from normal growth control involves an altered response to . Previous studies have indicated links between TGF-,B1-mediated GI growth arrest and the state of cyclins, CDKs, and the product of the retinoblastoma susceptibility gene, RB (9-13). Treatment of cells with TGF-p appears to prevent phosphorylation of RB and retains RB in the hypophosphorylated, growth-suppressive state. TGF-/31 also suppresses synthesis of CDK4, a major catalytic subunit of cyclin D1, in G1 in mink lung epithelial cells, and constitutive CDK4 synthesis in these cells leads to TGF-pl resistance (9). These results indicate that growth arrest by TGF-.81 might be mediated through the cyclins, CDKs, and RB-related pathways (9-13).To further investigate the function of cyclin D1 and its role in tumorigenesis, we have stably transfected an expression vector containing the human cyclin D1 cDNA in HET-1A cells, a human normal esophageal epithelial cell line immortalized by simian virus 40 (SV40) large tumor antigen CT antigen) (14). Oversynthesis (>10 fold) of cyclin D1 protein in clones of HET-1A cells led to an increase in colonyforming efficiency and saturation density. In addition, the clones were less responsive to growth inhibition by TGF-,81 than the parental cells or control vector cell clones. Interestingly, these clones which express increased amounts of cyclin D1 exhibited a decrease in the amount of TGF-(3 type II receptor.