Johannes L. Bos

Genetic alterations during colorectal-tumor development

Because most colorectal carcinomas appear to arise from adenomas, studies of different stages of colorectal neoplasia may shed light on the genetic alterations involved in tumor progression. We looked for four genetic alterations (ras-gene mutations and allelic deletions of chromosomes 5, 17, and 18) in 172 colorectal-tumor specimens representing various stages of neoplastic development. The specimens consisted of 40 predominantly early-stage adenomas from 7 patients with familial adenomatous polyposis, 40 adenomas (19 without associated foci of carcinoma and 21 with such foci) from 33 patients without familial polyposis, and 92 carcinomas resected from 89 patients. We found that ras-gene mutations occurred in 58 percent of adenomas larger than 1 cm and in 47 percent of carcinomas. However, ras mutations were found in only 9 percent of adenomas under 1 cm in size. Sequences on chromosome 5 that are linked to the gene for familial adenomatous polyposis were not lost in adenomas from the patients with polyposis but were lost in 29 to 35 percent of adenomas and carcinomas, respectively, from other patients. A specific region of chromosome 18 was deleted frequently in carcinomas (73 percent) and in advanced adenomas (47 percent) but only occasionally in earlier-stage adenomas (11 to 13 percent). Chromosome 17p sequences were usually lost only in carcinomas (75 percent). The four molecular alterations accumulated in a fashion that paralleled the clinical progression of tumors. These results are consistent with a model of colorectal tumorigenesis in which the steps required for the development of cancer often involve the mutational activation of an oncogene coupled with the loss of several genes that normally suppress tumorigenesis.

K-ras Oncogene Activation as a Prognostic Marker in Adenocarcinoma of the Lung

BACKGROUNDnThe capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas.nnnMETHODSnWe studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes.nnnRESULTSnNineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001).nnnCONCLUSIONSnThe presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.

The ras gene family and human carcinogenesis

It has been well established that specific alterations in members of the ras gene family, H-ras, K-ras and N-ras, can convert them into active oncogenes. These alterations are either point mutations occurring in either codon 12, 13 or 61 or, alternatively, a 5- to 50-fold amplification of the wild-type gene. Activated ras oncogenes have been found in a significant proportion of all tumors but the incidence varies considerably with the tumor type: it is relatively frequent (20-40%) in colorectal cancer and acute myeloid leukemia, but absent or present only rarely in, for example, breast tumors and stomach cancer. No correlation has been found, yet, between the presence of absence of an activated ras gene and the clinical or biological features of the malignancy. The activation of ras oncogenes is only one step in the multistep process of tumor formation. The presence of mutated ras genes in benign polyps of the colon indicates that activation can be an early event, possibly even the initiating event. However, it can also occur later in the course of carcinogenesis to initiate for instance the transition of a benign polyp of the colon into a malignant carcinoma or to convert a primary melanoma into a metastatic tumor. Apparently, the activation of ras genes is not an obligatory event but when it occurs it can contribute to both early and advanced stages of human carcinogenesis.

Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung.

To define the role of cellular oncogenes in human cancers, we studied the prevalence of mutational activation of ras oncogenes in untreated non-small-cell lung cancer. Genomic DNA was extracted from 39 tumor specimens obtained by thoracotomy and was examined for activating point mutations in codons 12, 13, and 61 of the H-ras, K-ras, and N-ras genes. A novel, highly sensitive assay based on oligonucleotide hybridization following an in vitro amplification step was employed. The K-ras gene was found to be activated by point mutations in codon 12 in 5 of 10 adenocarcinomas. Two of these tumors were less than 2 cm in size and had not metastasized. No ras gene mutations were observed in 15 squamous-cell carcinomas, 10 large-cell carcinomas, 1 carcinoid, 2 metastatic adenocarcinomas from primary tumors outside the lung, and 1 small-cell carcinoma. An approximately 20-fold amplification of the unmutated K-ras gene was observed in a tumor that proved to be a solitary lung metastasis of a rectal carcinoma. We conclude that mutational K-ras activation may be an important early event in the pathogenesis of adenocarcinoma of the lung but that amplification of ras genes or mutational activation of H-ras or N-ras does not play a major part in non-small-cell lung cancer.

Insulin stimulation of gene expression mediated by p21ras activation

In fibroblasts, insulin is a weak mitogen and does not induce expression of c‐fos, c‐jun or p33. However, increasing the expression levels of either normal p21Hras or the insulin receptor, but not mutant p21Hras, enables insulin to induce the expression of these genes. In cells expressing elevated levels of insulin receptor, this process involves a rapid increase in p21rasGTP levels (from 20% to 70% GTP as a percentage of total guanine nucleotides). No increase in p21rasGTP levels was observed after PDGF and EGF stimulation of cells expressing high levels of the cognate receptor, stressing the specificity of the insulin‐induced increase. We conclude that in fibroblasts, p21ras is an intermediate of the insulin signal transduction pathway involved in the regulation of gene expression and mitogenicity.

The 2.2 kb E1b mRNA of human Ad12 and Ad5 codes for two tumor antigens starting at different AUG triplets

By nucleotide sequence analysis and S1 nuclease mapping we have determined the structural organization of early region E1b of Ad12. We have also revised the nucleotide sequence of the E1b region of Ad5. Both regions have an identical structural organization and show considerable homology at the nucleotide level. The major tumor antigens (Ad12, 19 and 54 kilodaltons [kd]); Ad5, 21 and 55 kd) are encoded in two overlapping reading frames. A single mRNA of 2.2 kilobases codes for both these proteins, depending on which AUG triplet serves as the start codon: the 19-21 kd protein initiates at the 5-promximal AUG; the 54-55 kd protein initiates at the second AUG in another reading frame. Peptide mapping shows that the small and large tumor antigens do not share common tryptic peptides, in accordance with the nucleic acid sequence data. In addition, the 19-21 kd protein can also by synthesized from a one kilobase mRNA. Finally, the gene for the Ad12 analog of protein IX is characterized.

A novel function of the transforming domain of E1a : repression of AP-1 activity

Adenovirus E1a represses transcription of the collagenase gene via the phorbol ester-responsive element (collTRE). The mechanism involves inhibition of the trans-activating function of the transcription factor AP-1 without reduction of its synthesis and without any apparent change in DNA binding or composition. The ability of E1a to downmodulate AP-1 is a unique property among dominant oncogenes. This repression depends on conserved region 1, one of the transforming domains of E1a, indicating that it is an integral feature of adenovirus transformation.

Activated N-ras controls the transformed phenotype of HT1080 human fibrosarcoma cells

To investigate whether the activated N-ras oncogene of HT1080 human fibrosarcoma cells contributes to the expression of the transformed phenotype, we have isolated flat revertants. In two independent revertant lines, an increase in chromosomal ploidy occurred without a concomitant increase in the number of copies of the N-ras transforming allele. Immunoprecipitation confirms that the level of the mutant N-ras p21 gene product in the revertants is correspondingly lower than in HT1080. Analysis of sporadic tumors derived from the revertant cells reveals an increased dosage of the transforming allele. The revertants also retransform after transfection of cloned activated ras oncogenes. These results imply direct participation of an N-ras oncogene in maintaining the transformed phenotype of a human tumor cell line.

Differential effects of the adenovirus E1A oncogene on members of the AP-1 transcription factor family.

The adenovirus early region 1A (E1A) oncogene interferes with the expression level and activity of the AP-1 transcription factor family. E1A abolished the transactivating function of AP-1 (Jun/Fos), which binds to the 12-O-tetradecanoylphorbol-13-acetate-responsive element of the collagenase gene (collTRE). In contrast, the activity of another member of the AP-1 family that binds to the c-junTRE was not repressed. The mRNA expression of the c-jun gene was, in fact, strongly elevated in various cell types expressing the E1A gene of either adenovirus type 5 (Ad5) or Ad12. The regulation of the junB gene by adenovirus E1A, on the other hand, depended both on the cell type and on the transforming adenovirus serotype. The fact that E1A-induced alterations in the repertoire of AP-1 transcription factors depend on its transforming domain in conserved region 1 suggests that the effects are relevant for the transformation process.

Possible involvement of normal p21 H-ras in the insulin/insulinlike growth factor 1 signal transduction pathway.

Expression of a mutant H-ras gene confers a transformed phenotype to rat-1 fibroblasts which is basically independent of exogenous growth factors (GFs). Rat-1 cells induced to express high levels of the normal H-ras gene were also found to display a transformed phenotype. In contrast to cells expressing mutant H-ras, these cells were dependent on GFs. We used this difference in GF dependence to analyze a possible involvement of exogenous GFs in H-ras function. Compared with untransformed rat-1 cells, cells overexpressing normal H-ras displayed an elevated response toward insulinlike growth factor 1 (IGF-1), insulin, and bombesin and an increased sensitivity toward phosphatidic acids. It was found that 8-bromo-cyclic AMP inhibited the responses to all GFs in rat-1 cells but had no effect on mutant-H-ras-transformed cells. In cells overexpressing normal H-ras, 8-bromo-cyclic AMP inhibited the responses to all GFs except those to insulin and IGF-1. This implies that overexpression of normal H-ras in the presence of insulin/IGF-1 is functionally similar to the expression of mutant H-ras, since mutant H-ras can circumvent this block by itself. These and other results strongly suggest a functional linkage between insulin/IGF-1 and normal p21 H-ras.