Zanotti Silvia, Fisseler-Eckhoff Annette, Mannherz Hans Georg
Department of Anatomy and Embryology, Ruhr-Universität, Bochum, Germany.
Gynecol Oncol. 2003 Jun;89(3):376-84. doi: 10.1016/s0090-8258(03)00061-1.
We compared the capacity of cells in normal cervical epithelium, progressive stages of CIN, and invasive carcinoma to proliferate, differentiate, and undergo apoptosis.
We investigated 30 conizations showing regular squamous epithelium of the ectocervix, all stages of cervical preinvasive neoplastic lesions (CIN I to III), or invasive carcinoma. The expression of the cell proliferation and differentiation marker Ki67 and Mad-1, respectively, and of the apoptosis-related proteins bcl-2, active caspase-3, and DNase I was analyzed on paraffin sections by immunohistochemistry. The expression of DNase I or -like enzymes was also analyzed at the level of their gene transcripts by in situ hybridization. In addition, apoptotic events were identified by in situ end labeling of fragmented DNA (ISEL).
Expression of Ki67 was restricted to suprabasal cells in normal cervical epithelium but increased with CIN severity and invasive carcinoma. ISEL demonstrated apoptosis in superficial layers of normal, CIN I, and CIN II epithelium, whereas in CIS (CIN III) and invasive carcinoma, ISEL-positive cells were additionally observed at varying epithelial locations. Bcl-2 immunostaining remained restricted to the basal layer of all preneoplastic and neoplastic stages. Active caspase-3 was present in the suprabasal layer and extended to all upper layers in normal epithelium and slightly decreased with increasing dysplasia. In invasive carcinoma it was restricted to few scattered cells. The differentiation marker Mad-1 extended from the spinous to the superficial layer in regular epithelium, but gradually shifted to more superficial layers with increasing CIN grade and invasive carcinoma. A similar topological change was observed for DNase I with increasing CIN grade. In CIS and invasive carcinoma, DNase I immunopositive cells were solely interspersed within neoplastic cells. In contrast, DNase I specific mRNA was present in all epithelial layers in CIN III and neoplasia, suggesting a translational block of the expression of DNase I or -like enzymes.
Our data indicate that the elevated proliferation observed with increasing CIN severity and carcinoma was not paralleled by a similar increase in cell elimination. Most of the dysplastic and neoplastic cervical epithelial cells appeared incapable of entering terminal differentiation and complete it by apoptosis, possibly due to their failure to express or activate apoptosis executing enzymes.
我们比较了正常宫颈上皮、CIN进展期及浸润癌中细胞的增殖、分化及凋亡能力。
我们研究了30例宫颈锥切标本，其显示宫颈外口为规则的鳞状上皮、宫颈上皮内瘤变（CIN I至III）各期或浸润癌。通过免疫组织化学分析石蜡切片上细胞增殖和分化标志物Ki67和Mad-1以及凋亡相关蛋白bcl-2、活化的半胱天冬酶-3和脱氧核糖核酸酶I的表达。还通过原位杂交在基因转录水平分析脱氧核糖核酸酶I或类似酶的表达。此外，通过对断裂DNA的原位末端标记（ISEL）鉴定凋亡事件。
Ki67的表达在正常宫颈上皮中局限于基底上层细胞，但随CIN严重程度和浸润癌而增加。ISEL显示正常、CIN I和CIN II上皮表层有凋亡，而在原位癌（CIN III）和浸润癌中，ISEL阳性细胞在不同上皮位置额外可见。Bcl-2免疫染色在所有癌前和肿瘤阶段均局限于基底层。活化的半胱天冬酶-3存在于基底上层并延伸至正常上皮的所有上层，且随发育异常增加略有减少。在浸润癌中，它局限于少数散在细胞。分化标志物Mad-1在正常上皮中从棘层延伸至表层，但随CIN分级增加和浸润癌而逐渐向更表层转移。随着CIN分级增加，脱氧核糖核酸酶I也观察到类似的拓扑变化。在原位癌和浸润癌中，脱氧核糖核酸酶I免疫阳性细胞仅散布于肿瘤细胞内。相反，脱氧核糖核酸酶I特异性mRNA在CIN III和肿瘤的所有上皮层中均存在，提示脱氧核糖核酸酶I或类似酶的表达存在翻译阻断。
我们的数据表明，随着CIN严重程度和癌的增加所观察到的增殖升高并未伴随着细胞清除的类似增加。大多数发育异常和肿瘤性宫颈上皮细胞似乎无法进入终末分化并通过凋亡完成分化，这可能是由于它们未能表达或激活凋亡执行酶。
