Reduced binding activity of transcription factor NF-kappa B accounts for MHC class I repression in adenovirus type 12 E 1-transformed cells.

The early region 1 (E1) of human adenovirus (Ad) type 12 represses the expression of major histocompatibility (MHC) Class I genes in transformed primary rodent cells. In this paper we show that both NF-kappa B and KBF1 (p50 dimer) binding activity to the H2TF1 element in the Class I promoter is reduced in Ad12-13S-E1A-transformed cells compared to Ad5E1- or Ad12-12S-E1A-transformed cells. Consistently, in Ad12E1A-13S-transformed cells the H2TF1 element does not contribute to transcriptional activity in transient expression assays, whereas it does contribute in Ad12E1A-12S-transformed cells. Therefore, the most likely explanation is that reduced binding of NF-kappa B and KBF1 to the H2TF1 element accounts for the down-regulation of MHC Class I expression in Ad12E1- and Ad12E1A-13S-transformed cells.

Co-regulated expression of junB and MHC class I genes in adenovirus-transformed cells.

The expression of the junB gene parallels the expression of the MHC class I genes in Adenovirus (Ad) transformed cells. In Ad12E1-transformed primary BRK cells both genes are transcriptionally repressed only when the 13S product of Ad12E1A is present. This indicates that repression of MHC class I and junB genes is a function of conserved region 3 (CR3) of the Ad12E1A protein. In Ad5-transformed BRK cells expression of these genes is unchanged. In established NRK cells, however, introduction of Ad12E1A does not cause repression of the MHC class I and junB genes, but in these cells Ad5E1A increases the expression of both MHC class I and junB. Using mutant Ad5E1A genes, it is shown that this activation is mediated by CR1. Introduction of a functional junB gene under the control of a heterologous promoter in Ad12E1-transformed BRK cells causes no increase in MHC class I expression. This demonstrates that the down-regulation of junB is not directly responsible for class I repression, but rather that both genes are coregulated by the Ad12E1 region.

Ras oncogene mutations in basal cell carcinomas and squamous cell carcinomas of human skin.

Activated ras oncogenes have been detected in a variety of human malignancies. Activation of ras oncogenes usually occurs by point mutations within specific codons of the H-ras, N-ras, and K-ras genes. For the present study, DNA was isolated from 30 basal cell carcinomas (BCC) and 12 squamous cell carcinomas (SCC). After amplification of genomic DNA by using the polymerase chain reaction, the occurrence of point mutations was investigated with 32P-labeled synthetic oligonucleotides. These probes are complementary to the known point-mutated nucleotide sequences of the ras genes. In four out of the 30 BCC studied, point mutations were detected at codon 12 of the K-ras gene and at codon 61 of the H-ras gene. The K-ras mutations involve glycine to cysteine and glycine to asparagine amino acid changes. The mutation at codon 61 of the H-ras gene is consistent with a replacement of glutamine by histidine. In one SCC, a point mutation was detected at codon 12 of the K-ras gene, involving a glycine to cysteine substitution in the gene product. These findings demonstrate that mutational activation of ras genes takes place in skin carcinomas, but the rate at which these mutations occur seems to be relatively low.

Activated ras genes in human seminoma: evidence for tumor heterogeneity.

The incidence of mutations in cellular ras genes was determined in human seminoma, a germ cell tumor of the testis, with the aid of specific oligonucleotide probe hybridization. To eliminate the large number of nonneoplastic cells present in seminomas, aneuploid tumor cell nuclei were isolated from the tumor tissue by flow sorting. Mutations were detected in 40% of the seminomas at codons 12 or 61 of either the Ki-ras or the N-ras gene. No correlation was found with histopathological or clinical features. In some seminomas the mutant gene was present in only a fraction of the tumor cell population, suggesting tumor heterogeneity for ras gene mutations. Yet, flow cytometric measurement of nuclear DNA contents and histological examination of tumor tissue did not reveal two different tumor cell populations. We conclude from these observations that ras mutation is probably not the initial genetic event in the development of seminoma.

N-ras mutations in human cutaneous melanoma from sun-exposed body sites.

In 7 of 37 patients with cutaneous melanoma, mutations in the N-ras gene were found. The primary tumors of these seven patients were exclusively localized on body sites continuously exposed to sunlight. Moreover, the ras mutations were all at or near dipyrimidine sites known to be targets of UV damage. Two primary tumors were biclonal with respect to ras mutation. An active role for UV irradiation in induction of the mutations is suggested.

Mechanisms of ras mutation in myelodysplastic syndrome.

Using synthetic oligonucleotide hybridization, we have found a ras mutation in 11 of 27 patients (41%) with primary or non-therapy related myelodysplastic syndrome (MDS). This high incidence of mutation, mainly of the N-ras oncogene, was generally found in patients with disease progression to acute leukemia (8 of 11 patients = 73%). Two general mechanisms of ras mutation were found. In five patients the ras mutation was present in only a fraction of the cells; sometimes appearing in late stages of the disease, suggesting that it can occur in a differentiated cell clone. In six other patients, the ras mutation was present in the great majority of bone marrow or blood cells. The mutation was detected in mature normal lymphocytes and persisted following a complete clinical remission in two of these patients, implying that the ras mutation can occur in an early stage of cell differentiation or stem cell. Patients with a ras mutation had a median survival of nine months (all patients dead) compared to 16 patients without a ras mutation that had a median follow-up of 16 months (10 patients alive; P less than 0.005). Since 9 of the 11 patients (82%) with a ras mutation were found to have an abnormal monocytic component at diagnosis or during disease evolution, it is possible that in myeloid disorders a ras mutation is preferentially associated with myelomonocytic cell differentiation.

Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung.

47 tumor samples, 45 of which were obtained at thoracotomy for non-small cell lung cancer were examined for mutational activation of the oncogenes H-ras, K-ras, and N-ras. A novel, highly sensitive assay based on oligonucleotide hybridization following an in vitro amplification step was employed. ras gene mutations were present in nine of 35 adenocarcinomas of the lung (all K-ras), in two of two lung metastases of colorectal adenocarcinomas (1 x K-ras, 1 x N-ras) and in one adenocarcinoma sample obtained at autopsy (H-ras). All K-ras and H-ras mutations were in either position 1 or 2 of codon 12, while the N-ras mutation was in position 2 of codon 61. The potential clinical significance of K-ras activation was analyzed using the combined results of this and of our earlier study (S. Rodenhuis et al., New Engl. J. Med., 317: 929-935, 1987). Lung adenocarcinomas with K-ras mutations tended to be smaller and were less likely to have spread to regional lymph nodes at presentation. With a median follow up of 10 months, survival data are still immature. None of six adenocarcinomas of nonsmokers had a K-ras mutation and only one of four who had stopped smoking more than 5 years before. We conclude that mutational K-ras activation is present in about a third of adenocarcinomas of the lung and that the mutational event may be a direct result of one or more carcinogenic ingredients of tobacco smoke. Studies involving larger numbers of patients are required to confirm the association of K-ras activation with smoking and the inverse relation with tumor progression.

KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas.

DNAs from human pancreatic adenocarcinomas were analyzed for the presence of mutations in codons 12, 13 and 61 of the NRAS, KRAS and HRAS gene. Formalin-fixed and paraffin-embedded tissue was used directly in an in vitro amplification reaction to expand the relevant RAS sequences. The mutations were detected by selective hybridization using mutation-specific synthetic oligonucleotides. In 28 of the 30 patients we found a mutation in codon 12 of the KRAS gene. This result confirms the findings of Almoguera et al. [Cell 53 (1988) 549-554] that KRAS mutations occur frequently in adenocarcinomas of the exocrine pancreas. The mutations are predominantly G-T transversions, in contrast to the KRAS mutations in colon tumors which are mainly G-A transitions. Furthermore, in a portion of the tumors the mutation appears to be homozygous.