Genetic Analysis of A Familial Adenomatous Polyposis Pedigree / 胃肠病学

Background: The adenomatous polyposis coli (APC) gene mutation is closely related to the risk of familial adenomatous polyposis (FAP). Aims: To analyze the genetic etiology of a FAP pedigree. Methods: FAP was diagnosed in a patient with repeated hematochezia after the receiving of endoscopy, pathological analysis and family history investigation. Genetic analysis was conducted by new-generation sequencing (NGS) technique in the FAP family members. Results: A heterozygous mutation of APC gene c.2800_2803delACTT (p.T934fs) was existed in the family members, which led to the frame shifts of APC gene and resulted in the change of encoding amino acids sequence and formed the abnormal proteins. This mutation could lead to serious FAP symptoms with early onset of colorectal adenoma and carcinogenesis. Conclusions: NGS can diagnose the disease much earlier and more accurately, and provide a new detection method for the early prevention of genetic diseases. The heterozygous mutation of p.T934fs is the root etiology of the clinical phenotype of this FAP pedigree, and polypectomy should be considered in affected members for preventing the carcinogenesis of colon.

Cdc20 mutation promotes development of colon cancer in APCmin/+ mice / 肿瘤研究与临床

Objective To demonstrate the relationship between Cdc20 mutation and the promotion of colon cancer via Cdc20loxp/+ APCmin/+ villin-cre+/-compound mutant mice.Methods Cdc20loxp/+ APCmin/+ villin-cre+/-compound mutant mice and APCmin/+ mutant mice were generated by mice mating strategy.The colon tumors of two group mice were compared by phenotypic analysis and histology analysis.Results Phenotypic analysis showed that the number of tumors in Cdc20loxp/+ APCmin/+ villin-cre+/-compound mutant mice group and APCmin/+ mutant mice group was 1.2±0.5 and 1.6±0.5, respectively (t =0.215, P =0.588), and the maximum diameter of tumors was (2.7±0.3) cm and (2.5±0.2) cm, respectively (t =0.568, P =0.575).Pathologic type of Cdc20loxp/+ APCmin/+ villin-cre+/-compound mutant mice was adenocarcinoma, while that of APCmin/+ mice was tubular adenoma.Conclusion Cdc20 carrying a null allele can accelerate the promotion of colon cancer in APCmin/+ mice without influence on the tumor number and size.

APC protein immunoexpression in colorectal adenoma and adenocarcinoma / Imunoexpressão da proteína APC nos tecidos de adenoma e de adenocarcinoma colorretais

BACKGROUND: activation of the Wnt pathway by mutated APC gene is considered the initial event in colorectal carcinogenesis. The identification of these mutations can improve the specific treatment of the adenocarcinoma. OBJECTIVE: detect and evaluate wild-type APC protein in tissue from colorectal adenoma, adenocarcinoma and adjacent mucosa. METHODS: 42 patients that underwent surgery for adenocarcinoma and 53 patients with resected adenomas were studied. Tissue samples from the adenocarcinoma were obtained from the tumor and from adjacent non-neoplastic mucosa located 10 cm from the proximal margin of the tumor. Adenoma tissue was obtained from representative areas. Blocks of tissue microarray (TMA) were submitted to immunohistochemistry with anti-APC, with readings of positivity and intensity of immunostaining and the score of immune expression of APC protein was obtained. RESULTS: the APC protein immune expression score showed a significantly lower expression of APC protein in the adenoma when compared with the adenocarcinoma (p < 0.0001) and adjacent mucosa (p < 0.0001). The APC protein immune expression score in the colorectal mucosa and adjacent to the adenocarcinoma showed no significant difference (p = 0.24). CONCLUSIONS: the finding of decreased expression of APC protein in adenoma tissue may indicate that the mutated APC gene may contribute to the changes in the adenoma-carcinoma process of carcinogenesis sequence. The strong expression of protein APC in tissues from the carcinoma and adjacent mucosa suggests that in most patients in this series, the mutation of the APC gene did not participate in the oncogenesis mechanism. (AU)

RACIONAL: a ativação da via Wnt pelo gene APC mutado é considerado evento inicial da carcinogênese colorretal. A identificação dessas mutações pode tornar o tratamento do adenocarcinoma mais específico. OBJETIVO: detectar e avaliar a proteína APC não mutada em tecidos de adenoma, adenocarcinoma e mucosa adjacente. MÉTODO: estudados 42 doentes operados de adenocarcinoma e 53 com adenomas ressecados. Tecidos de adenocarcinoma foram obtidas da neoplasia e da mucosa adjacente não neoplásica situadas a 10 cm da margem proximal do tumor. Tecidos do adenoma foram obtidas de área representativa. Blocos de tissue microarray (TMA) foram submetidos a imuno-histoquímica com anticorpo anti-APC. Avaliadas a positividade e intensidade da expressão e obtidos escores da imunoexpressão da proteína APC. RESULTADOS: o escore da imunoexpressão da proteína APC no adenoma foi significantemente menor do que no adenocarcinoma (p < 0,0001) e na mucosa adjacente (p < 0,0001). O escore da imunoexpressão da proteína APC na mucosa adjacente e no adenocarcinoma não mostraram diferença significante (p = 0,24). CONCLUSÕES: a menor expressão da proteína APC no adenoma pode indicar que o gene APC mutado participa das alterações do processo adenoma-carcinoma. A forte expressão da proteína APC no CCR e na mucosa adjacente sugerem que a mutação do gene APC não participou da oncogênese. (AU)

Construction of pEGFP-N3-APC vectors carrying various APC functional domains and their expression in HCT-II6 cells / 肿瘤研究与临床

Objective To construct recombinant plasmids containing various functional domains of APC protein and detect their expression in HCT-116 cells. Methods Five APC gene fragments were amplified by PCR with whole APC gene as template and primers designed according to APC cDNA sequence and mutation cluster domain. The five obtained fragments were cloned into eukaryotic expression vector pEGFP-N3 to generate recombinant pEGFP-N3-APC1-5. Sequence of the inserted gene was identified and analyzed after restriction enzyme digestion. Liposome-mediated recombinant plasmid pEGFP-N3-APC was transfected into HCT 116 cells and identified by green fluorescence. RT-PCR was employed to validate the expression of recombinant vectors in cells. Results Recombinant pEGFP-N3-APC1-5 were confirmed by restriction enzyme digestion and sequence analysis. The plasmids could be expressed in HCT-116 cell line detected by fluorescence microscope. Results of RT-PCR made clear that vectors constructed could be expressed in HCT-116 cells. Conclusion The relative efficient expression of five recombinant expressive vector in HCT-116 cell line may provide an experimental basis for selecting specific therapy peptide for colorectal cancer.

Experimental study on triptolide reversing hypermethylation of APC in acute lymphoblastic leukemia cell line Jurkat / 白血病·淋巴瘤

Objective To observe the effects of triptolide(TPL) on the anti-oncogene-APC gene of acute lymphoblastic leukemia cell line Jurkat in vitro. Methods The effects of TPL on proliferation Jurkat cells were assayed by using cell culture, MTT. The effects of TPL on APC gene of Jurkat cells were analyzed by nested methylation specific PCR and RT-PCR. The effects of TPL on the proteinum expression of APC gene were detected by Western blotting analysis. Results Following the treatment of TPL, the cell proliferation rate was degraded as the treatment concentration increased and the culture time extended. The effects were dose and time-dependent. The 48 hour IC50 was 19.7 ng/ml. TPL can reverse hypermethylation of APC gene,and induce the expression of the mRNA and the proteinum. Conclusion Low dose TPL could depress the proliferation rate of Jurkat. The possible mechanism might be its reversing the hypermethylation of APC gene and activiting the expression of APC gene.