Elsevier

Gynecologic Oncology

Volume 89, Issue 3, June 2003, Pages 376-384
Gynecologic Oncology

Regular article
Changes in the topological expression of markers of differentiation and apoptosis in defined stages of human cervical dysplasia and carcinoma

https://doi.org/10.1016/S0090-8258(03)00061-1Get rights and content

Abstract

Objective

We compared the capacity of cells in normal cervical epithelium, progressive stages of CIN, and invasive carcinoma to proliferate, differentiate, and undergo apoptosis.

Methods

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).

Results

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.

Conclusions

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.

Introduction

Cervical cancer is the second most common cancer among women worldwide. The vast majority of carcinomas originate within the transformation zone of the cervix uteri, where proliferating cells are exposed at the squamocolumnar junction [1]. CIN (cervical intraepithelial neoplasia) is characterized by abnormal cellular proliferation, abnormal maturation as manifested by loss of polarity, cellular disorganization, and nuclear atypia, and the level to which mitotic figures are present [2], [3], [4], [5], [6], [7]. The CIN grading classifies these lesions into grades I, II, or III, corresponding to mild, moderate, or severe dysplasia/carcinoma in situ [5]. In many cases, dysplastic areas (CIN I and II) regress spontaneously [7]. CIN III is regarded as being identical to carcinoma in situ (CIS), from which an invasive cervical carcinoma may develop in about 50% [6], [7].

The severity of dysplastic alterations found within patches of the cervical epithelium correlates with clinical prognosis, i.e., the probability to progress into cervical carcinoma. It is still a matter of debate whether the “dysplastic” cell is already a tumor cell. Dysplastic cells appear to be unable to initiate terminal differentiation; they possess highly variable morphologies and fail to attain the layer-specific cell shape. Therefore, additional parameters are needed to further classify the alterations in dysplastic cells.

In multicellular organisms, the tissue cell number is held constant by equal rates of cell proliferation and elimination. Physiologically, cells are eliminated by programmed cell death, which in most cases is executed in the morphologically and biochemically defined form of apoptosis. Morphologically, apoptosis is characterized by condensation of the cytoplasm and the cell nucleus followed by their fragmentation and final phagocytotic elimination by macrophages or neighboring cells [8], [9], [10], [11], [12]. In recent years a number of gene products have been identified that regulate the initiation of apoptosis by activation of apoptosis executing enzymes like the caspases (cyteinyl aspartate proteases) [see 13] and endonucleases [14], [15], [16], [17]. In humans, 11 different caspases have been identified, some of which function as signaling and others as executing caspases, in particular caspase-3 [13]. Caspases reside in resting cells as inactive zymogens, but after initiation of apoptosis are activated by proteolysis by signaling caspases or autocatalytically [13]. Ca2+/Mg2+-dependent endonuclease(s) catalyzes chromatin degradation first into large fragments and finally at internucleosomal sites [see 14]. In most instances, the apoptotic endonucleases are generated by the dying cell itself (cell autonomously); however, there are also indications that chromatin degradation of dying cells may be catalyzed or at least completed by DNases of phagocytotic cells or extracellular endonucleases [18], [19]. A number of candidate apoptotic endonucleases have been suggested, such as the caspase activated DNase initially isolated from lymphatic cells [16], the expression of which in humans is, however, restricted to a few tissues [20]. Therefore, different endonucleases might catalyze apoptotic chromatin degradation in other tissues. Deoxyribonuclease I (DNase I) has been proposed as another candidate enzyme [17]; however, DNase I deficient mice recently generated in our laboratory have indicated that DNase I is more likely involved in extracellular chromatin clearance or that of necrotic cells [19 and unpublished data]. DNase I like enzymes have been identified which exhibit about 60% sequence identity to DNase I and possess almost identical enzymatic properties. Furthermore, they were shown to be intracellularly expressed and to possess a nucleus localization signal [21], [22], [23]. These enzymes were identified in rat (DNase γ or DNaseY) and humans (DNAS1L3 or nhDNase) and at least DNase γ was shown to participate in the cell autonomous DNA cleavage during apoptosis [21], [22], [23].

Tumor growth can be due to an increased rate of proliferation or a decreased rate of cell elimination. Here we attempted to histochemically evaluate this equilibrium in normal cervical epithelium, progressive stages of CIN, and invasive carcinomas with the aim to identify and correlate possible disturbances of this equilibrium with increasing stages of neoplastic severity. It is hoped that such an analysis might be helpful in the development of novel strategies of treatment of cervical carcinoma or of prognostic indicators for the response to radio- or chemotherapy.

Section snippets

Materials

We purchased the primary polyclonal (pAb) antibodies against the differentiation marker Mad-1 (Santa Cruz, Heidelberg/Germany), the active form of caspase-3 (Molecular Probes Europe, Leiden/The Netherlands), and monoclonal antibodies against the nuclear proliferation marker Ki67 (Mib-1; Dianova, Hamburg/Germany) and the apoptosis regulating gene product Bcl-2 (Dako, Hamburg/Germany). The generation of the polyclonal antibodies against purified mouse deoxyribonuclease I have been described

Immunostaining for marker proteins of proliferation and differentiation

First we analyzed by immunostaining the expression of Ki67 and Mad-1, as marker for proliferation and differentiation, respectively. In regular cervical epithelium only a few nuclei of para- or suprabasal cells were stained by the monoclonal antibody MiB-1 against the nuclear antigen Ki67 (Fig. 1A). The basal cells were negative in normal cervical epithelium (Fig. 1A) and in all CIN stages and CIS (Fig. 1B–D). In invasive squamous cell carcinoma almost all cells of the surface epithelium and

Discussion

It is well established that cervical carcinogenesis develops after progression through a number of definable preinvasive neoplastic lesions that are characterized by grade specific morphological alterations. Cervical carcinomas and the precursor grades CIN I–III are associated with infections by human papilloma virus (HPV) [34]. Epidemiological and in vitro studies have shown that the high-risk HPV types, 16,18, and 31, code for proteins that strongly influence cell cycle and genome stability

Acknowledgements

We thank Mrs. K. Klar for technical help and Dr. A. Ricken (Bochum) for critically reading the manuscript. This work was financially supported by the Medical Faculty of the Ruhr-University Bochum (FoRUM) and the Deutsche Forschungsgemeinschaft, Bonn, Germany.

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    Present address. Institute of Pathology and Cytology, HSK-Wiesbaden, D-65199 Wiesbaden, Germany.

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