A New Hypothesis Of Carcinogenesis And Its Primary Examination

What is the cause of cancer is still a matter of debate. On the bases of various hypothesizes of carcinogenesis we proposed a better model for understanding the process of carcinogenesis. The model proposed that cancer is a disease of the expansion of cancer stem cells, which is accompanied by cycle of immortal DNA strand establishment and amplification and random distribution, which is induced by gene mutation and aneuploidy.Aim: To test our proposal of carcinogengsis and aneuploidy hypothesis and the prevention of symmetric division of stem cells hypothesis respectively.Methods: 1) Initiation and promotion two-stage chemical carcinogenesis method was used to induce cutaneous tumors in the mouse back skin. DNA label retaining method was used to chase the lapse of BrdU positive cells in mouse skin and tumor tissues and to predict what was happen to immortal DNA strands in the process of carcinogenesis. DNA ploidy image analysis was performed to detect the karyotype of tumor cells. DNA fluorescence in situ hybridization was used to test the length changes of telomeres in the process of carcinogenesis. Immunolabelling assay was uesd to analysis where were Integrin β1, Integrin α6, Keratin 19 and Keratin 15 expressed in the tumor tissues and to predict whether the label retaining cells in the tumor tissueswere tumor stem cells. All the methods mentioned above were used to test our proposal of carcinogengsis.2) Plate clonality assay and regular chromosome assays were respectively used to test the proliferation potential and karyotypes of BT325> 786-CK 293 > C6 and NIH3T3 cell lines. The relationship between proliferation phenotype and karyotype of tumors was used to test aneuploidy hypothesis.3) Plate clonality assays was used to test the impact of cell density to cell clonality and cell cycle in BT325> 786-CK 293 > C6 and NIH3T3 cell lines. The relationship berween the impact of cell density to cell clonality and cell cycle was used to test the prevention of symmetric division of stem cells hypothesis.Results: 1) There were 32 mice got papillomas, averaging 1-3 papillomas/mouse three-four months after the initial tumor induction. During the chase period, immunolabeling results showed that an extremely high percentage of keratinocytes and cutaneous tumor cells incorporated the BrdU at 24 h after the final BrdU injection. Some keratinocytes of mice labeled with BrdU during the juvenile period were BrdU positive for at least 60 days after the final BrdU injection. Most of these cells, which can be stimulated to divide by treatment with TPA and result in near complete loss within 10 days, were located within hair follicle and some were scattered throughout the basal layer of the skin. In contrast, no keratinocytes labeled with BrdU during the adult period were BrdU positive at 30 days after the final BrdU injection. However, BrdU label-retaining cells could be produced in the adult mice skin by TPA treatment 3 days before BrdU labeling. BrdU positive cells were observed in tumor tissues for at least 30 days but not longer than 60 days after the final BrdU injection. Based on the measure of DNA content, DNA ploidy imageanalysis showed that all induced tumor cells were aneuploid. DNA fluorescence in situ hybridization results showed that the telomere fluorescence intensity of normal skin was more positive than that of TPA treated skin and papillomas tissue. Telomere fluorescence signal was positive in the primary epidermal culture cells but not in the uterine cervix cancer tissues of 7 cases and Hela and C6 cells. The karyotypes assays show that Hela and C6 were aneuploid. HE staining showed that the anaphase bridges were easily observed in Hela and C6 cells. Plate clonality assays showed that the rate of clonogenic cells in DMB A and/or TPA treated epidermal was more than that in normal epidermal. Immunolabelling assay showed that Integrin pi, Integrin oc6 and Keratin 15 were mainly expressed in the hair follicles of normal and TPA treated skin but not papilloma tissues. Keratin 19 was positive in the hair follicles of normal and TPA treated skin and the base line of papilloma tissues. Keratin 10 was positive in the suprabasal cell layer of normal and papilloma tissues. PCNA was rare expressed in the normal skin but TPA treated skin and papilloma tissues.2) The karyotypes assays show that the karyptype of all these cell lines are aneuploidy and their model number are 32-41, 47-57, 58-68, 50-59, 58-68 , the ration of cells which chromosome number is in the model number are 72.6%, 49%, 61.2%, 73.4% and 67.3% respectively. The fraction of colony-forming cells in BT325> 786-0 ^ 293, C6 and NIH3T3 cell lines is 15%, 12%, 13%, 13% and 15% respectively. Of the BT325 cells clonality studied, 8% had the greatest reproductive capacity and 17% intermediate growth potential and 75% least growth potential. These three type cells produced holoclones, meroclones and paraclones respectively. The progressive conversion of holoclone to meroclone to paraclone was normally a...