APC and K-ras gene mutation in aberrant crypt foci of human colon

AIM:To study the genetic alteration in ACF and to define the possibility that ACF may be a very early morphological lesion with molecular changes,and to explore the relationship between ACF and colorectal adenoma even carcinoma. METHODS: DNA from 35 CRC, 15 adenomas, 34 ACF and 10 normal mucus was isolated by means of microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well as the mutation cluster region (MCR) of APC gene was performed. RESULTS: K-ras gene mutation frequency in ACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/15), and 14.3% (5/35) respectively, showing no difference (P 】 0.05) in K-ras gene mutation among three pathologic procedures. The K-ras gene mutation in adenoma, carcinoma and 4 ACF restricted in codon 12 (GGT GAT), but the other 2 mutations from ACF located in codon 13 (GGC GAC). K-ras gene mutation was found more frequently in older patients and patients with polypoid cancer. No mutation in codon 61 was found in the three tissue types. Mutation rate of APC gene in adenoma and carcinoma was 22.9% (8/35) and 26.7% (4/15), which was higher than ACF (2.9%) (P 【0.05). APC gene mutation in carcinoma was not correlated with age of patients, location, size and differentiation of tumor. CONCLUSION: ACF might be a very early morphological lesion in the tumorogenesis of colorectal tumor. The morphological feature and gene mutation status was different in ACF and adenoma. ACF is possibly putative microadenoma that might be the precursor of adenoma. In addition, the development of a subgroup of colorectal carcinomas might undergo a way of normal epithelium ACF carcinomas .

Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.

Preliminary Investigation on Regulating Effects of Different TCM Treatments on Transcription of the Correlated Genes of Liver Cancer in Rats

The regulating effects of TCM treatments including clearing away heat and toxic materials,promoting blood circulation and removing blood stasis,and strengthening the spleen and regulating qi on the oncogene transcription were observed in the liver cancer model rats.The preliminary results indicated that the mRNA levels of H-ras N-ras and K-ras,and signal molecules correlated with the ras/MAPK signal transduction pathway were down-regulated by the different TCM treatments in varying degrees.Also,the regulating effects of the treatments on differently-displayed genes were discrepant.It is suggested that the molecular mechanisms of the TCM treatments for liver cancer was complex with different target genes.

Detecting K-ras and p53 gene mutation from stool and pancreatic juice for diagnosis of early pancreatic cancer

OBJECTIVE: To explore new methods for the early diagnosis of pancreatic cancer through detection of K-ras and p53 mutations in pancreatic juice and stool. METHODS: 201 patients in PUMC Hospital from 1994 - 2000 and 60 control individuals were enrolled in this study. K-ras point mutation was detected by PCR-RFLP while p53 mutation was detected by PCR-SSCP. RESULTS: K-ras mutation was found in pancreatic juice in 87.8% (36/41) of pancreatic cancer patients and 23.5% (4/17) of benign pancreatic disease patients. In 261 stool specimens, amplification found mutations successfully in 235 patients (90%). K-ras mutation was found in stool in 88% (66/75) of pancreatic cancer patients, 51.1% (24/47) of benign pancreatic disease patients and 19.6% (9/46) of normal individuals. p53 mutation was found in pancreatic juice in 47.4% (18/38) of pancreatic cancer patients and 12.5% (2/16) of benign pancreatic disease patients. p53 mutation was found in stool in 37.1% (23/62) and 19.1% (4/21) of chronic pancreatitis patients. CONCLUSION: K-ras mutation in pancreatic juice has higher diagnosis sensitivity and specificity, and therefore may be used as a supplement in the diagnosis of pancreatic cancer. Detection of K-ras mutation combined with p53 mutation in stool can aid in the screening of pancreatic cancer.

Effect of blocking Ras signaling pathway with K-Ras siRNA on apoptosis in esophageal squamous carcinoma cells

OBJECTIVE： To study the effect of RNAi silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells. METHODS： EC9706 cells were treated in the follow- ing six groups： blank group （no transfection）, nega- tive control group （transfection no-carrier）, trans- fection group （transfected with pSilencer-siK-ras）, taxol chemotherapy group, taxol chemotherapy plus no-carrier group, taxol chemotherapy plus transfection group. Immunocytochemistry, Reverse transcription-polymerase chain reaction and west- ern blotting were used to analyze the expression of MAPK1 （mitogen-activated protein kinases 1） and cyclin D1 in response to siRNA （small interfering RNA） transfection and taxol treatment. RESULTS： K-Ras （K-Ras gene） siRNA transfection of EC9706 esophageal squamous carcinoma cells de- creased the expression of K-Ras, MAPK1 and cyclinD1 at the mRNA and protein level. Reverse tran- scription-polymerase chain reaction indicated that the expression levels of MAPK1 and cyclin D1 mRNAs were significantly lower in the transfection group than in the blank group （P〈0.05）. Western blotting showed that 72 h after EC9706 cell trans- fection, the expression levels of MAPK1 and cyclin D1 proteins had decreased in all groups, and the ex- pression levels in the transfection group were sig- nificantly inhibited as compared with the blank group. Apoptosis increased significantly in the transfection group or after addition of taxol as com- pared with the blank group and the no-carrier group. The degree of apoptosis in the taxol plus transfection group was more severe. CONCLUSION： Apoptosis increased significantly in EC9706 esophageal carcinoma cells after siRNA-me- diated inhibition of Ras signaling, with the most ob- vious increase observed in the transfection plus tax- ol chemotherapy group. Ras knockdown therefore increased cellular sensitivity to the chemotherapeu- tic agent, taxol. Ras knockdown also down-regulat- ed the expression of the downstream genes, MAPKI and cyclin DI, thus inhibiting the growth, proliferation and metabolism of esophageal cancer cells.

Zebularine对人鼻咽癌细胞CNE-2Z增殖的抑制作用

某些抑癌基因转录起始区域的启动子发生异常超甲基化，会导致抑癌基因表达失活引发肿瘤，因此有关去甲基化药物的研究正在成为肿瘤治疗领域的热点。在国外研究中已证实抗肿瘤药——Zebularine（Zeb）可以抑制多种肿瘤细胞生长，并上调因甲基化失活的相应抑癌基因表达。但Zeb对鼻咽癌细胞的影响在国内、外还未见报道，鉴于鼻咽癌在我国华南地区的高发率，我们对此进行初步探讨，以期为鼻咽癌的综合治疗提供新的思路。