Bianchi A B, Navone N M, Aldaz C M, Conti C J
Department of Carcinogenesis, University of Texas, M.D. Anderson Cancer Center, Smithville 78957.
Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7590-4. doi: 10.1073/pnas.88.17.7590.
A significant role for mouse chromosome 7 abnormalities during chemically induced skin carcinogenesis has been advanced based on previous cytogenetic and molecular studies. To determine the frequency of allelic losses at different loci of chromosome 7 in skin tumors induced in the outbred SENCAR mouse stock by a two-stage initiation-promotion protocol, we compared the constitutional and tumor genotypes of premalignant papillomas and squamous cell carcinomas for loss of heterozygosity at different informative loci. In a previous study, these tumors had been analyzed for their allelic composition at the Harvey ras-1 (Ha-ras-1) locus and it was found that 39% of squamous cell carcinomas had lost the normal Ha-ras-1 allele exhibiting 3 or 2 copies of the mutated counterpart or gene amplification. In the present study, by combining Southern blot and polymerase chain reaction fragment length polymorphism analyses, we detected complete loss of heterozygosity at the beta-globin (Hbb) locus, distal to Ha-ras-1, in 15 of 20 (75%) skin carcinomas. In addition, 5 of 5 informative cases attained homozygosity at the int-2 locus, 27 centimorgans distal to Hbb. Polymerase chain reaction analysis of DNA extracted from papillomas devoid of stromal contamination by fluorescence-activated sorting of single cell dispersions immunolabeled with anti-keratin 13 antibody revealed loss of heterozygosity at the Hbb locus, demonstrating that this event occurs during premalignant stages of tumor development. Interestingly, loss of heterozygosity was only detected in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion. Analysis of allelic ratios by densitometric scanning of tumors that had become homozygous at Hbb but retained heterozygosis at Ha-ras-1 indicated mitotic recombination as the mechanism underlying loss of heterozygosity on mouse chromosome 7 during chemically induced skin carcinogenesis. These findings are consistent with the presence of a putative tumor suppressor gene linked to the Hbb locus in the 7F1-ter region of mouse chromosome 7, the functional inactivation of which may constitute a critical event in skin tumor progression, possibly during the malignant conversion stage.
基于先前的细胞遗传学和分子研究，已提出小鼠7号染色体异常在化学诱导的皮肤癌发生过程中起重要作用。为了确定远交系SENCAR小鼠品系经两阶段启动-促进方案诱导产生的皮肤肿瘤中7号染色体不同位点的等位基因缺失频率，我们比较了癌前乳头瘤和鳞状细胞癌的组成型和肿瘤基因型，以检测不同信息位点的杂合性缺失情况。在先前的一项研究中，已对这些肿瘤的哈维ras-1（Ha-ras-1）位点的等位基因组成进行了分析，发现39%的鳞状细胞癌丢失了正常的Ha-ras-1等位基因，呈现出3个或2个拷贝的突变对应物或基因扩增。在本研究中，通过结合Southern印迹和聚合酶链反应片段长度多态性分析，我们在20例皮肤癌中的15例（75%）中检测到Ha-ras-1远端的β-珠蛋白（Hbb）位点完全杂合性缺失。此外，在5例信息性病例中，有5例在Hbb远端27厘摩的int-2位点达到纯合性。通过对用抗角蛋白13抗体免疫标记的单细胞分散体进行荧光激活分选，从无基质污染的乳头瘤中提取DNA进行聚合酶链反应分析，结果显示Hbb位点存在杂合性缺失，表明该事件发生在肿瘤发展的癌前阶段。有趣的是，仅在表现出高度发育异常和微侵袭区域的晚期病变中检测到杂合性缺失。通过对在Hbb位点已变为纯合但在Ha-ras-1位点仍保留杂合性的肿瘤进行光密度扫描分析等位基因比例，结果表明有丝分裂重组是化学诱导的皮肤癌发生过程中小鼠7号染色体杂合性缺失的潜在机制。这些发现与小鼠7号染色体7F1-ter区域中与Hbb位点相关的一个假定肿瘤抑制基因的存在相一致，该基因的功能失活可能是皮肤肿瘤进展中的一个关键事件，可能发生在恶性转化阶段。
