Regular articleChanges in the topological expression of markers of differentiation and apoptosis in defined stages of human cervical dysplasia and carcinoma
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.
References (55)
Apoptosis
Immunol Today
(1993)- et al.
Cell deaththe significance of apoptosis
Int Rev Cytol
(1980) - et al.
The central executioners of apoptosiscaspases or mitochondria?
Trends Cell Biol
(1998) - et al.
The apoptosis endonucleasescleaning up after cell death
Trends Cell Biol
(1994) Human papillomaviruses in the pathogenesis of anogenital cancer
Virology
(1991)- et al.
Expression of bcl-2 oncoprotein in gastrointestinal and uterine carcinomas and their premalignant lesions
Hum Pathol
(1997) - et al.
Expression of bcl-2 expression in invasive and in situ carcinoma of the uterine cervix
Am J Obstet Gynecol
(1998) - et al.
Caspasesmore than just killers?
Trends Immunol
(2001) - et al.
Apoptosis related proteins in cervical intraepithelial neoplasia and squamous cell carcinoma of the cervix
Gynecol Oncol
(2002) - et al.
A novel single amino acid deletion caspase-8 mutant in cancer cells that lost proapoptotic activity
J Biol Chem
(2002)
Time-lapse cinematographic observations of normal human cervical, dysplasia, and carcinoma in situ
J Natl Cancer Inst
Definition of precursors
Gynecol Oncol
The three group metaphase as morphologic indicator of high ploidy cells in cervical intraepithelial neoplasia
Anal Quant Cytol Histol
Natural history of cervical intraepithelial neoplasia
Clin Obstet Gynecol
Carcinoma and other tumors of the cervix
Cervical cancer detection in British Columbia. A progress report
J Obstet Gynaecol Br Commonw
Progression and regression of cervical lesionsreview of smears from women followed without initial biopsy or treatment
J Clin Pathol
Apoptosisa basic biological phenomenon with wide-ranging implications in tissue kinetics
Br J Cancer
Apoptosismolecular mechanisms in programmed cell death
Eur J Cell Biol
Apoptosismolecular regulation of cell death
Eur J Biochem
New aspects of the mechanism of DNA fragmentation in apoptosis
Biochem Cell Biol
A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD
Nature
Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death)
EMBO J
An auxillary mode of apoptotic DNA fragmentation provided by phagocytes
Genes Dev
Features of systemic lupus erythematosus (SLE) in Dnase 1 deficient mice
Nat Genet
Molecular cloning and characterizytion of human caspase-activated DNase
Proc Natl Acad Sci USA
Identification of an endonuclease responsible for apoptosis in rat thymocytes
Eur J Biochem
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Present address. Institute of Pathology and Cytology, HSK-Wiesbaden, D-65199 Wiesbaden, Germany.