Jun Miyoshi

K-Ras is essential for the development of the mouse embryo

ras genes encode members of the small GTP-binding proteins. Ras protein is highly conserved in various species from yeast to humans and plays a key role in signal transduction. Ras is related to cell proliferation and differentiation. While, in addition, mutations in the ras genes are implicated in a variety of tumors. However, the physiological functions and specific roles of each ras gene, H-ras, K-ras and N-ras, are still not fully understood. To clarify the role of the K-Ras in vivo, we generated K-ras mutant mice by gene targeting. In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term. At embryonic day 15.5, their ventricular walls are extremely thin. Besides, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord. Our results thus indicate K-Ras to be essential for normal development in mice and residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice.

Structure and transforming potential of the human cot oncogene encoding a putative protein kinase.

A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3 end in the last coding exon, which probably resulted in a deletion and an altered C terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor.

Differences in Effects of Oncogenes on Resistance to γ Rays, Ultraviolet Light, and Heat Shock

The effects of viral or activated cellular oncogenes on sensitivity to gamma rays, ultraviolet light, and heat shock were examined in SHOK (Syrian hamster Osaka-Kanazawa) cells and their transfectants. Resistance to gamma rays was conferred by the introduction of v-mos or c-cot genes, which coded serine/threonine kinase. Cells transfected with v-mos and c-cot genes increased their resistance to ultraviolet light and heat shock compared to their parent cells (SHOK cells). Of the activated ras genes, the N-ras gene developed a SHOK cell phenotype resistant to gamma rays and ultraviolet light. The Ha-ras gene produced SHOK cells resistant to ultraviolet light and heat shock, while introduction of the Ki-ras gene did not affect sensitivity. The v-erbB gene was found to be involved in the development of resistance to heat shock. Transfection with neo, c-myc, and v-fgr genes had little or no effect on cell survival. The karyotypes of SHOK cells and oncogene-containing cells were compared. No alterations were seen after the introduction of a foreign gene. Using cell cycle analysis, we found no apparent difference between SHOK cells and their transfectants. These results suggest that activation of serine/threonine kinase may be involved in common processes occurring after gamma-ray, ultraviolet-light, and heat-shock treatment, and that each oncogene may have a different effect on the development of a resistant phenotype.

A Simple and Useful Method for Simultaneous Screening of Elevated Levels of Expression of a Variety of Oncogenes in Malignant Cells

Abnormal expression of various oncogenes has been implicated in the development of many malignant tumors. Although RNA blotting methods have been used to measure abnormal expression, they involve the time‐consuming process of individually labeling the oncogene probes. To simplify this process we have attempted to develop a new method, termed simultaneous screening, which is based on the synthesis of radiolabeled cDNA corresponding to the mRNA population of malignant cells and on hybridization with various oncogene probes, immobilized on a membrane filter. This method circumvents the time‐consuming process of the prevailing RNA blotting methods and is also sensitive enough to detect accurately a five‐ to ten‐fold level of expression of rare mRNA (∼10 copies per cell). Overexpression of ten oncogenes was detected in a variety of malignant cells and mitogen‐stimulated cells with this method. These results suggest that our simultaneous screening method can be used to examine the overexpression of oncogenes.

A novel point mutation at codon 146 of the K-ras gene in a human colorectal cancer identified by the polymerase chain reaction

In this report, point mutations of the K-ras gene at codon 146 were analyzed in 25 cases of colon cancer, 4 cases of lung cancer, and 41 cases of lymphoid malignancy. A codon 146 mutation substituting threonine (ACA) for alanine (GCA) was detected in the tumor tissue of a patient with colon cancer and was not detected in the normal tissue of the same patient. Any additional mutations of theras gene family were not detected in this patient. These results suggest that the codon 146 mutation of the K-ras gene could be involved in the development of naturally occurring human malignancies.

The Murine cot Proto‐oncogene: Genome Structure and Tissue‐specific Expression

We cloned and analyzed the murine cot proto‐oncogene and examined its tissue‐specific expression in fetal, newborn and adult mice. Genomic cot DNA consists of eight exons, spanning more than 25 kb, and all intron‐exon borders are well conserved as compared to the human homolog. Analysis of the full‐length cot cDNA revealed that it contained an open reading frame of 1,401 nucleotides, like human cot proto‐oncogene. The sequence identity between murine and human cot gene is 84.4% at the nucleotide level and 93.9% at the deduced amino acid level. On northern blot analysis of poly (A)+ RNA, the cot message was detected at 2.9 kb in size. Expression of the cot gene was observed in many tissues from fetal to adult mice, though the level of expression was low in all tissues examined.

Function-dependent cooperation between oncogene activation and nonrandom chromosome change during tumorigenic conversion of Syrian hamster cells☆

We determined the tumorigenicity of SHOK cells, a Syrian hamster cell line, morphologically transformed by the introduction of either c-Ha-ras, c-Ki-ras, N-ras, v-mos, v-fgr, or v-src genes. When 1 x 10(6) cells were injected subcutaneously into nude mice, SHOK cells containing one of those oncogenes gave rise to tumors within 3 weeks, while the parental SHOK cells, or SHOK cells containing the neo gene, did not show tumorigenicity even 200 days after injection. From karyotype analysis, the addition of chromosome 3 was consistently found in tumor cells derived from SHOK cells containing the c-Ha-ras, c-Ki-ras, or v-mos genes, and the acquisition of chromosome 9 was a common change observed in tumor cells derived from SHOK cells morphologically transformed by the v-fgr or v-src gene. No consistent chromosomal change was observed in tumor cells containing the N-ras gene. The results obtained from Southern blot analysis showed that amplification of introduced oncogenes was detected in the case of c-Ha-ras, c-Ki-ras, N-ras, v-fgr, and v-src genes. These results indicate that nonrandom chromosome changes, along with the amplification of introduced oncogenes, are required for the tumorigenic conversion. Our findings propose the possibility that secondary chromosomal changes associated with the tumorigenic conversion of morphologically transformed cells occur dependent on the function of the primary activated oncogene introduced.

Cot: Cot on cogene product (vertebrates)

The chapter discusses the Cot proto-oncogene product that is a cytosolic serine/threonine PK and is implicated in signal transduction by growth factors. Two activated forms of COT oncogenes have been detected by transfection of SHOK cells with tumor DNAs extracted from human thyroid and colon carcinoma. Both genes were oncogenically activated during the transfection process because of truncation of the C-terminal domain. Seven coding exons were identified in the human and mouse cot locus. The COT and CT12COT oncogenes are rearranged in exon 6 and the intron between exon 6 and 7, respectively. With regard to the domain structure, the N-terminal region may be involved in the stability of Cot protein. As both the oncogenically activated versions truncated in the C-terminal region have shown constitutive kinase activities, it seems likely that the C-terminal region is involved in negative regulation of the protein kinase activity.