Ha-ras is a member of a multigene family in man which encode highly related proteins of 189 amino acids (p21). In vitro, ras proteins bind GTP, and p21 mutants with treonine at position 59 autophosphorylate at that residue. Mutation (at amino acids 12 or 61) and elevated expression of ras genes result in cell transformation in culture, and are also observed in many types of human tumours. Normal and mutant transforming ras proteins show no differences in localization, lipidation or GTP binding. However, mutations at position 12 in recombinant (Thr 59) p21 molecules were observed to affect autophosphorylation. We have expressed the full-length normal and T24 transforming (Gly----Val at position 12) Ha-ras proteins in Escherichia coli and have purified them to homogeneity (ref. 19 and M.G. et al., in preparation); these proteins bound GTP with approximately molar stoichiometry and with an affinity comparable to partially purified mammalian proteins. Microinjection of the T24 protein into quiescent rodent fibroblasts resulted in a rapid alteration in cell morphology, stimulation of DNA synthesis and cell division; in contrast, little response was observed with the normal protein. We now report that the normal ras protein has an intrinsic GTPase activity, yielding GDP and Pi. In contrast, the T24 transforming protein is reduced 10-fold in this activity. We suggest that this deficiency in GTPase is the probable cause for the transforming phenotype of the T24 protein.

Wnt proteins form a family of highly conserved, secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis. Wnt genes and Wnt signaling are also implicated in cancer. It has been shown that Wnt proteins bind to receptors of the frizzled family on the cell surface. Through several cytoplasmic relay components including DVL-1, the human counterpart of the Drosophila disheveled gene, the signal is transduced to β β β β-catenin, which then enters the nucleus and forms a complex with T-cell factor (TCF) to activate transcription of Wnt target genes. nt genes encode a family of highly conserved, secreted glycoproteins that modulate cell fate and behavior in embryos through activation of receptor-mediated signaling pathways. In the absence of a Wnt signal, β-catenin levels are kept low through interactions with the protein kinase zw3/ GSK-3β (zeste white-3, shaggy in Drosophila/glycogen synthase kinase-3β), CK1α (casein kinase 1α), APC (adenomatous polyposis coli) and axin.

To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Haras-) gene derived from the human T24 bladder carcinoma cell line and compared them Tumors are classically defined as benign or malignant (1). Benign tumors are noninvasive growths that do not spread to distant organs. Unless located in a functionally vital site (e.g., brain), they pose little threat to the patient and usually can be removed surgically. In contrast, malignant neoplasms are readily invasive, metastasize to organs throughout the body, and eventually kill their host (1). The biochemical events that distinguish malignant from benign neoplasms remain unidentified. Work in several laboratories has implicated oncogene activation in the expression of tumorigenicity but the role of oncogenes in the pathogenesis of malignant versus benign tumors has received little attention. The NIH/3T3 cell line, although immortalized in vitro, is reportedly nontumorigenic and is widely used as a recipient cell line for detecting transforming gene sequences isolated from tumorigenic cell lines or tissues (2-6). For example, upon transfection with the activated ras gene, NIH/3T3 cells form foci in tissue culture, exhibit anchorage independence, and, in those experiments in which in vivo studies have been conducted, produce tumors in nude mice (6-10). However, the behavior of the tumors formed by transfected NIH/3T3 cells has not been rigorously evaluated and it remains unclear whether oncogene activation is an event associated strictly with the pathogenesis of benign neoplasms or whether activation is also an essential feature for expression of metastatic properties. We report that transfection with the activated c-Ha-ras-l gene accelerates the tumorigenicity and enhances the metastatic potential of NIH/3T3 Tumorigenicity and Metastasis. Tumorigenicity and spontaneous metastatic potential were assayed by inoculating mice with different cell doses in the footpad (i.m.) or the supraclavicular region (s.c.). Tumor size was monitored at the supraclavicular site every 2-3 days by caliper measurement. For studies on experimental metastasis, different numbers of cells were injected into the tail vein of nude mice. At autopsy the major organs of all animals were examined both grossly and histologically for evidence of metastases. Single sections were prepared from each organ except the lung, in which case multiple sections were examined.Detection of Activated c-Ha-ras Oncogene and Human Alu Sequences. For preparation of DNA (12), cell monolayers established from primary tumors or metastatic foci were dispersed into phosphate-buffered saline (Pi/NaCl), pelleted, rinsed, resuspended in 10 mM Tris HCl, pH 8.0/0.35 M NaCl/1 mM EDTA, lysed in 0.5% NaDodSO4, and treated for 4-12 hr with Pronase (0.1 mg/ml) at 37°C. DNA was extracted with phenol, ethanol precipitated, and dissolved in 10 mM Tris-HCl, pH ...

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The prevalence of BRCA1/2 germline mutations in Japanese patients suspected to have hereditary breast/ovarian cancer was examined by a multi-institutional study, aiming at the clinical application of total sequencing analysis and validation of assay sensitivity in Japanese people using a cross-sectional approach based on genetic factors estimated from personal and family histories. One hundred and thirty-five subjects were referred to the genetic counseling clinics and enrolled in the study. Full sequencing analysis of the BRCA1/2 gene showed 28 types of deleterious mutations in 36 subjects (26.7%), including 13 types of BRCA1 mutations in 17 subjects (12.6%) and 15 types of BRCA2 mutations in 19 subjects (14.1%). Subjects were classified into five groups and 22 subgroups according to their personal and family history of breast and/or ovarian cancer, and the prevalence of deleterious mutations was compared with previously reported data in non-Ashkenazi individuals. Statistical analysis using the Mantel-Haenszel test for groups I through IV revealed that the prevalence of Japanese subjects was significantly higher than that of non-Ashkenazi individuals (P = 0.005, odds ratio 1.87, 95% confidence interval 1.22-2.88). Family history of the probands suffering from breast cancer indicated risk factors for the presence of deleterious mutations of BRCA1/2 as follows: (1) I n Japan, breast cancer is the most frequent malignancy in women and estimates of new cases and deaths in 2002 were 32 245 and 9178, respectively.(1) The standardized incidence ratio of breast cancer in Japan was approximately one-third that of the US (32.7 vs 101.7 per 100 000 women).(1) The incidence of breast cancer in Japanese women shows a steady increase; however, it is still much lower than in Western countries. In breast cancer, family history is the strongest risk factor for cancer predisposition. Epidemiological studies showed that 12% of women with breast cancer have one affected family member and 1% have two or more affected relatives.(2) Women with one, two, and three or more first-degree affected relatives have an increased breast cancer risk when compared with women who do not have an affected relative (risk ratios 1.8, 2.9, and 3.9, respectively).(2) Recent advances in molecular genetics elucidated BRCA1 and BRCA2 (BRCA1/2) as two major susceptibility genes for breast cancer predisposition.(3,4) Gene testing of BRCA1/2 is available as a routine clinical test for diagnosing hereditary breast/ovarian cancer (HBOC) in the US and other Western countries, (5,6) while only a few reports have been published concerning the prevalence of BRCA1/2 mutations among Japanese people. (7)(8)(9)(10)(11)(12) The methods of genetic analysis employed in these studies varied, such as polymerase chain reaction (PCR)/ single strand conformational polymorphisms (SSCP), protein truncation test, and PCR/direct sequencing, but they were performed as preliminary in-house tests in the research setting. In the US, commercial BRCA1/2 gene testing was initiated by M...

We investigated associations among intake of folate, vitamin B2, vitamin B6, vitamin B12, and polymorphisms of 5,10-methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) genes and breast cancer risk in a Japanese population. A hospital based, case-control study was conducted in Nagano Prefecture, Japan, in 388 pairs of patients with histologically confirmed invasive breast cancer and age- and area-matched controls selected from medical checkup examinees. Energy-adjusted intakes of folate and other B vitamins were derived from a validated food frequency questionnaire. Genotyping was completed for MTHFR (C677T and A1298T) and MTR (A2756G). Odds ratios and 95% confidence intervals were calculated by the conditional logistical regression model. Median dietary folate intake (microg/day) in the control group was 438.2 (interquartile range: 354.9-542.9). Neither dietary intake of folate, vitamin B2, vitamin B6, or vitamin B12 nor polymorphisms of MTHFR or MTR genes were significantly associated with breast cancer risk. Further, no significant interaction was found among nutrients, polymorphisms, and breast cancer risk. Associations of nutrients with breast cancer risk did not differ by hormone receptors status. We conclude that dietary intake of folate and related B vitamins and genotypes of MTHFR or MTR have no overall association with breast cancer risk in Japanese women.

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The hereditary breast and ovarian cancer (HBOC) registration system of Japan was established by the Japanese HBOC Consortium. The first trial was registered in 2015 in four institutions to which some registration committee members belonged. We analyzed the information of 830 Japanese pedigrees, who underwent BRCA1/2 genetic testing, including mutation carriers with BRCA1 (N = 127) and BRCA2 (N = 115), and their families. The mutation-positive rate was 19.7%. Variants of uncertain significance were found in 6.5% of all individuals subjected to genetic testing for BRCA1/2. Compared to the United States, Japan had a higher mutation-positive rate in most categories, except for the groups with male breast cancer. Among the intrinsic subtypes of BRCA1-associated breast cancers, 75.8% were triple-negative. The incidence rate of contralateral breast cancer in BRCA1/2 mutation carriers was 0.99%/year. Among 240 mutation carriers, 26 and 62 patients underwent risk-reducing mastectomy (RRM) and risk-reducing salpingo-oophorectomy (RRSO), respectively; the respective frequencies of occult cancer were 7.1 and 3.2%. Metachronous breast cancer after RRM or peritoneal cancer after RRSO was not observed during the follow-up period. The nationwide registration system began last year and the system enables followup analysis in Japan.