Activated c-Ha-ras oncogene with a guanine to thymine transversion at the twelfth codon in a human stomach cancer cell line.

The rat fibroblast cell line Rat 1 was transfected with total DNA of a gastrocarcinoma cell line, BGC-823. The transforming gene was cloned from the genomic library of the secondary transformants using in situ hybridization with a probe of the human Alu repeat sequence. This cloned gene is homologous to the protooncogene c-Ha-ras. The activation lesion of the transforming gene was identified by sequence analysis as a single nucleotide substitution of thymine for guanine in the 12th codon. This results in the substitution of valine for glycine at the 12th amino acid of the Mr 21,000 protein.

[Diagnosis of gastric carcinoma with a group of biochemical tests].

Results of five non-specific reactions (ceruloplasmin assay, Ehrlich's reagent colorimetry, heat-resisting reaction of serum and determination of indole and neutral sulphide in urea) for the diagnosis of gastric carcinoma were calculated by the use of multiple-factor analysis. The positive rates in 284 cases of gastric carcinoma (including 8 early lesions), 306 healthy donors and 1018 cases of benign gastric diseases were 78.2% (62.5% for the early gastric cancers), 0.7% and 15.7%, respectively. The results showed that these tests are valuable for the screening in a high incidence area of gastric cancer or as a supplementary diagnostic means of gastric carcinoma in clinical practice.

[Amplification and rearrangement of proto-oncogene c-abl in human leukemia cells].

DNA of peripheral blood or bone marrow leukocytes from 8 normal subjects, 7 cases of acute lymphocytic leukemia (ALL), 2 of acute myelogenous leukemia (AML) and 1 of chronic myelogenous leukemia (CML), having been digested by endonuclease Eco RI or Pst I separately, was hybridized with the probes of 3' fragment (Pst I/Hind III) or 5' fragment (Hinc II/Pst I) of Abelson murine leukemia virus (A-MuLV) oncogene v-abl. The proto-oncogene c-abl, which is homologous to v-abl, was found amplified in 4 ALL, 1 CML and 1 AML. In one of these 4 ALL, c-abl was amplified even over 100 times. A new c-abl BamH I fragment with 6.7 kilobase pairs (kb) in length was observed in 2 ALL and 1 CML out of these 6 cases with amplification, but none of this fragment was found in the normal subjects or other leukemia patients. These 3 patients with the presence of 6.7 kb fragment were high risk ones and 2 of them had died, suggesting that 6.7 kb fragment be the index of poor prognosis. The amplification and rearrangement of c-abl imply the activation of proto-oncogene in leukemogenesis.

[DNA transformation activity of a human gastrocarcinoma cell line].

DNAs of three cell lines of human gastrocarcinoma (MGC-803, BGC-823 and PACM-82) and two fresh solid tumors of human stomach cancer were used to transfect NIH3T3 and Rat-1 cells. The transformed cells were selected with high concentration of glucose and low concentration of serum, or with medium containing Geneticin (G418) after co-transfection of pSVneo and DNAs of stomach cancer cell line or primary transformants. From the second round transfection, we had obtained transformants which could grow with high colony forming efficiency in soft agarose and were tumorigenic in nude mice. The southern blot analysis showed that the cellular DNA of the transformants contained human Alu repeat sequence and the transformed gene from stomach cancer cell line (BGC-823) and was homologous to proto-oncogene c-Ha-ras. The transforming gene is able to induce neoplastic transformation of NIH3T3 and Rat-1 cells.

[Relation of c-Ha-ras point mutation to the prognosis and metastasis of gastric carcinoma].

Mouse fibroblast cell line NIH 3T3 was transfected with oncogene c-Ha-ras containing mutation at the 12th codon, which was cloned from a gastric cancer cell line. After the transformants were injected subcutaneously into the nude mice, metastatic foci in the lung were found. The exogenous c-Ha-ras and its transcript were identified in the cells of the metastatic foci. This shows that the expression of the mutated c-Ha-ras is responsible for the acquisition of the metastatic phenotype for the cells. Furthermore, PCR-RFLP analysis was used to determine the point mutation of c-Ha-ras in gastric cancer. 11 of 27 fresh tissues and cell lines from gastric cancer were identified to contain the mutation at the 12th codon, and a correlation was found between the point mutation and metastasis to the distant organs and the survival time after surgical operation.

[Loss of heterozygosity at different chromosomes in patients with breast cancer].

The loss of heterozygosity (LOH) at chromosomes 3p24, 7q31, 16q24.3 and 17p13.1 was measured in 15 patients with breast cancer by means of polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) or PCR-CA repeat analysis. In 36%, 36%, 50% and 56% of the informative cases, LOH was detected at 3p24, 7q31, 16q24.3 and 17p13.1 loci, respectively. All the LOH at 3p24 or 7q31 happened in stage II or III, 67% of the LOH at 3p24 or 7q31 happened in the cases with lymph node metastasis, and 67% of the LOH at 3p24 or 7q31 were found in estrogen receptor negative patients. This finding shows that the measurement of LOH at 3p24 and 7q31 may be an useful predicting marker for the prognosis of patients with breast cancer. The high percent of LOH at these four loci may also indicate the possibility of the existence of tumor suppressor genes for breast cancer.

[Amplification of oncogenes HER2, mdm-2 and myc in breast cancer determined by modified competitive PCR].

The amplification of oncogenes HER2, mdm-2 and myc was determined with a modified competitive PCR. The HER2, mdm-2 and myc genes were found to amplify in 5, 6 and 6 out of 15 patients with breast cancer respectively. The amplification of HER2 and mdm-2 correlated with advanced tumors and lymph node involvement respectively, indicating that the amplification of the oncogenes is a useful indicator for the prognosis of patients with breast cancer and for the study of the biological behaviors of tumors. This modified non-radioactive competitive PCR is simple to handle (even for DNA samples with low quantity and low quality), and accurate for measuring the copy numbers of genes.

The antisense c-Ha-ras oligodeoxynucleotides inhibit the proliferation of the gastrocarcinoma-DNA-transformed rat 3-3 cells and reduce the p21 expression of the cells.

The Rat 3-3 is a secondary transformant of the rat fibroblast cell line (Rat-1) transfected with total DNA of a gastrocarcinoma cell line BGC-823. The cells over-express the c-Ha-ras oncogene which contains point mutation at the 12th codon. In order to determine how the activated c-Ha-ras oncogene expression governs the cell's transformation, two pentadecadeoxy-nucleotides AS-1 and AS-2 were synthesized. AS-1 was complementary to the single strand of the first three codons and the upstream sequence close to the ribosome binding site of c-Ha-ras mRNA. AS-2 was complementary to the 3' end of the first intron and the 5' end of the second exon of c-Ha-ras unripe RNA enclosed in the nucleus. The oligonucleotides could block either the translation of c-Ha-ras mRNA or the splicing of c-Ha-ras unripe RNA, thus inhibiting the expression of the activated c-Ha-ras oncogene and the proliferation of the transformed cells Rat 3-3. The inhibitory effect increased with a growing concentration of the antisense oligodeoxynucleotide (from 4-10 mumol/L) and reached its peak at 12 h after Rat 3-3 was treated with AS-1 and AS-2. This effect became weaker afterwards. The p21 level, product of c-Ha-ras, in the Rat 3-3, was examined by ELISA method. The result shows that the amount of p21 in the growth-inhibited cells is about 30% of that of the control cells.

Inhibited neoplastic phenotype by the c-Ha-ras antisense RNA.

A 2.0 kb fragment DNA plasmid which expresses antisense to the upstream first exon of c-Ha-ras oncogene was transfected into Ha-ras transformed cell lines, GCM-3T3 and REF-4.3. The transfection leads to the inhibition of malignant behaviour, shown by decreasing of growth speed, colony forming ability on soft agar, tumorigenicity in nude mice and increasing of differentiation degree. In GCM-3T3 cells the lung metastasis frequency became much less (from 60% to 12.5%) after the transfection and the expression of ras oncogene product, and p21 protein was obviously decreased in the transfected cells. This work has first shown the inhibitory effect of antisense RNA on neoplastic behaviour in China.

Correlation of mutations of oncogene C-Ha-ras at codon 12 with metastasis and survival of gastric cancer patients.

Mutations of c-Ha-ras at codon 12 were detected from 11 out of 27 fresh tissues and cell-lines of human gastric cancer patients using PCR-restriction analysis. Further statistical investigation showed that the presence of point mutations was related to the distal metastases and the survival time of gastric cancer patients after surgical operations.

Molecular cloning and nucleotide sequence analysis of a transforming gene from human gastrocarcinoma cell line.

Mouse and rat fibroblasts were transfected with total DNA from human gastrocarcinoma cell line BGC-823. It was shown by hybridization assay that the genome of one of the rat secondary foci contains transforming genes from the human gastrocarcinoma cell line, which are homologous to the protooncogene c-Ha-ras in the normal cells. The genomic library of the rat secondary foci was constructed, using lambda phage EMBL3 as the vector. The transforming gene Ha-ras of the human gastrocarcinoma cell was thus cloned by screening the library with the probes of human Alu repeat sequence and c-Ha-ras. The nucleotide sequences of the first and second exons were analysed by M13-dideoxy method. The result shows that the nucleotide sequence of the transforming gene is the same as that of the normal protooncogene except one nucleotide difference in the first exon.