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[Cancer Research 61, 7118-7121, October 1, 2001]
© 2001 American Association for Cancer Research

Carcinogenesis

Evidence for a Putative Senescence Gene Locus within the Chromosomal Region 10p14–p151

Manuela Poignée, Claudia Backsch, Katrin Beer, Lars Jansen, Nikola Wagenbach, Eric J. Stanbridge, Richard Kirchmayr, Achim Schneider and Matthias Dürst2

Gynäkologische Molekularbiologie, Abteilung Frauenheilkunde, Frauenklinik der Friedrich-Schiller-Universität Jena, 07743 Jena Germany [C. B., K. B., L. J., A. S., M. D.]; Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany [M. P., N. W.]; Gynäkologische Praxis, 89073 Ulm, Germany [R. K.]; and Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697-4025 [E. J. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High-risk human papillomavirus (HPV) types 16 and 18 are involved in the multistep process of cervical cancer. Transfection of normal keratinocytes with high-risk HPV-DNA generally gives rise to immortal cultures. This may be explained by the loss of senescence genes as a consequence of HPV-induced genetic instability. On the basis of the dominance of cellular senescence over immortality, fusion of normal keratinocytes with HPV-immortalized cells results in complementation of these putative gene defects. In a previous study, we showed that underrepresentation of chromosome 10 is a characteristic phenomenon during the early phase of immortalization. Here we show that introduction of a normal copy of chromosome 10 into HPV16-immortalized cells (HPKII) by Microcell-mediated chromosome transfer resulted in senescence of a significant number of hybrids. By using several derivatives of chromosome 10 for further fusion experiments, the chromosomal region responsible for senescence could be assigned to 10p14–p15. The potential significance of loss of gene function in this region is underlined by the high frequency (38.7%) of loss of heterozygosity in cervical cancers including early stage tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High-risk HPVs3 such as HPV16 and HPV18 are causative agents for high-grade intraepithelial neoplasia (CIN3) and cervical cancer. However, epidemiological data and experimental studies demonstrate clearly that infection per se does not suffice to induce malignancy. One mechanism by which the virus contributes to disease progression is by causing genetic instability of the host genome (1 , 2) . This is explained in part by the interaction of the viral oncoproteins E6 and E7 with key cell regulatory proteins such as p53 and pRb thereby deregulating the cell cycle, cell differentiation, DNA repair, and apoptosis (3, 4, 5, 6, 7) .

Cell culture experiments have shown that transfection of high-risk HPV DNA into keratinocytes increases the proliferative life span of the cells and in most cases yields immortal clones (8 , 9) . This property is not shared with low-risk types such as HPV6 and HPV11. We have shown in previous studies that several chromosomal aberrations occur during the early phase of immortalization. Comparative genomic hybridization revealed a characteristic underrepresentation of chromosome 4 and chromosome 10 in HPV16- or HPV18-transfected keratinocytes from different individuals (10) . Because it is known from somatic cell fusion experiments that the immortal phenotype of cells is recessive (11 , 12) , these chromosomes may be considered as likely candidates for the location of senescence genes.

In independent experiments, we introduced by Microcell-mediated chromosome transfer the entire human chromosome 10 and several derivatives of chromosome 10 into HPV16-immortalized human keratinocytes (HPKII) and monitored the hybrids for senescence. Using this functional approach, we could map a putative senescence gene locus to the distal part of the short arm of chromosome 10. Moreover, we could show by LOH analysis that this particular region is deleted frequently in cervical carcinomas.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Microcell-mediated Chromosome Transfer.
Micronucleation and Microcell fusion were performed as described previously (13) .

Cell Lines.
The Microcell donor cells contained an entire copy of human chromosome 10 (MCH941.6) or a derivative chromosome 10 (XIV10.2-1a, 17.I-3, and 17.IV-3), each tagged with a neomycin resistance gene (Table 1)Citation . For each fusion experiment, HPV16 immortalized cells (HPKII) served as recipients. All cells were grown in DMEM supplemented with 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. HPKII x chromosome 10 hybrids were selected in the presence of 400 µg/ml G418.


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  		Table 1 Summary of all cell lines used in this study

 
Biopsy Material.
Frozen biopsy specimens of 20 immature metaplasias, 5 low-grade cervical intraepithelial lesions (CIN1), 24 high-grade cervical intraepithelial lesions (CIN2 and CIN3), and 32 squamous cervical carcinomas were investigated. From each tissue, areas consisting predominantly of epithelial cells (metaplasia), dysplastic cells (CIN), and tumor cells (cancers) were prepared by manual microdissection using a Zeiss micromanipulator. For guidance, 5-µm thin sections adjacent to a 20-µm thick section required for microdissection were stained with H&E. The quality of microdissection was confirmed by staining and microscopic evaluation. Microdissected tissues (103 to 104 cells) were each placed in 25 µl [10 mM Tris, 50 mM KCl (pH 8.0), and 0.1% Tween 20]. Proteinase K was added to a final concentration of 100 µg/ml, and the reaction mix was incubated for 1 h at 56°C. Proteinase K digestion was repeated once more. The reaction was stopped by heating for 10 min at 96°C, followed by storage at -80°C until use.

Microsatellite PCR with Chromosome 10 Polymorphic Markers.
Twenty-five ng of genomic DNA extracted from blood or cultured cells (NucleoSpin columns; Machery-Nagel) or 1 µl of the cell suspension (crude DNA extract) obtained from microdissected tissue was used for microsatellite PCR. A total of 27 highly polymorphic repetitive sequences located on chromosome 10 (listed in Fig. 4Citation ) were analyzed. Sequence information for primers was obtained from the genome database (4) . The reaction mix for PCR consisted of 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates each, 4 pmol of each primer (one of which was labeled with IRD800 at its 5' end), and 0.4 unit of Tfl DNA polymerase (Biozym) in a volume of 25 µl. The reaction conditions included an initial denaturation step for 3 min at 94°C, followed by 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 58°C, and elongation for 1.5 min at 72°C. The last elongation step was performed for 3 min at 72°C. The microtiter plate was wrapped in aluminum foil and stored at -20°C until analysis. The PCR products were separated in 7% Long Ranger gels in a semiautomated sequencing apparatus equipped with a fluorescent detection system (LICOR, Michael Weichselgartner).



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  		Fig. 4. Partial deletion maps of human chromosome 10 of all donor cell lines by microsatellite PCR. • and {circ}, presence or absence of loci, respectively.

 
FISH.
FISH analysis was performed with digoxigenin labeled Coatasome 10 probe, according to standard protocol (Oncor).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of Senescence after Transfer of a Normal Copy of Chromosome 10 into HPKII Cells.
In three independent Microcell-mediated chromosome transfer experiments, a total of 16 HPKII x MCH941.6 cell hybrid colonies were marked on the culture dishes for individual monitoring during all phases of expansion. Of these, four hybrids could not be expanded beyond 12 pd. Hybrids (10-7) senesced after 20 pd. Senescent hybrids typically showed an increased cytoplasmic/nuclear ratio and cell flattening reminiscent of aging human primary keratinocyte cultures. For most of the senescent hybrids, sufficient cells were available for microsatellite PCR to confirm the presence of the introduced chromosome. All of the remaining hybrids could be cultured continuously beyond 50 pd. The presence of the transferred chromosome was confirmed by FISH analysis (Figs. 1Citation and 2Citation ) and by microsatellite PCR. In comparison with HPKII parental cells, most of the nonsenescent hybrids showed reduced rates of proliferation (Fig. 3)Citation . Moreover, several hybrids showed morphological signs of differentiation. Only two hybrids (11.II-1 and 11.III-1) showed no phenotypical alterations when compared with parental HPKII cells.



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  		Fig. 1. FISH analysis of HPKII parental cells using Coatasome 10 as a hybridization probe and a CyTM3-labeled antibody. The metaphase is counterstained with 4',6diamidino-2-phenylindole. The short arm of chromosome 10 is underrepresented.

 


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  		Fig. 2. FISH analyses of hybrids 14.II-1 (a), 11.II-1 (b); 8.II-1 (c) derived from HPKII x MCH941.6 fusions, and 10.V-3 (d) derived from the HPKII x XIV10.2-1a fusion. Each hybrid contains the introduced chromosome 10. For hybrid 8.II-1, the transferred chromosome is partially deleted.

 


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  		Fig. 3. Rates of proliferation of HPKII parental cells and of three hybrid cell clones (8.II-1, 14.II-1, and 14.III-1) derived from HPKII x MCH941.6 fusions. HPKII cells have a mean doubling time of 43 h, whereas the hybrids 8.II-1, 14.II-1, and 14.III-1 have mean doubling times of 135, 134, and 89 h, respectively.

 
Establishing New Donor Cell Lines with Derivatives of Chromosome 10.
To map the senescence gene locus on chromosome 10, we generated three new donor cell lines, each containing a particular derivative of chromosome 10 in the same mouse genetic background. For this purpose, Microcells were prepared from two different HPKII x MCH941.6 hybrids for fusion with mouse A9 cells. One der (10) was isolated from a hybrid (10, 9, 8, 7) , which showed extremely slow proliferation and senescence at 20 pd. The resulting donor line is referred to as XIV10.2-1a. One further der (10) was isolated from a hybrid (11.II-1) that showed no phenotypic alteration when compared with parental HPKII cells. From these fusions, two new donor lines were established and referred to as 17.I-3 and 17.IV-3. FISH analyses showed that all three donor cell lines stably retained the human derivatives of chromosome 10. The cell lines were characterized by microsatellite PCR using 27 polymorphic marker for chromosome 10 (Fig. 4)Citation . Donor cell lines 17.I-3 and 17.IV-3 appear to be identical.

Mapping a Putative Senescence Gene Locus to a Region within 10p14–p15.
Further Microcell fusion experiments were conducted with the above-described new donor cells. As for the fusions with the entire chromosome 10, 20 of 57 (35%) of the HPKII x XIV10.2-1a hybrids senesced before reaching 12 pd. In contrast, most HPKII x 17.I-3 and HPKII x 17.IV-3 hybrids could be expanded beyond 50 pd. The data are summarized in Table 2Citation .


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  		Table 2 Summary of the percentage of hybrids obtained after fusion

 
On the basis of the assumption that each hybrid can be scored individually and culture conditions were constant for all fusion experiments, the observed difference in the induction of senescent hybrids is statistically highly significant (P < 0,01) for: (a) HPKII x MCH941.6 and HPKII x 17.I-3/17.IV-3; and (b) HPKII x XIV10.2-1a and HPKII x 17.I-3/17.IV-3 fusions (Fisher’s exact test). Therefore, by comparing the deletion map of chromosome 10 of each donor cell line (Fig. 4)Citation , it can be deduced that the putative senescence gene locus must be located on the short arm of chromosome 10 in the region of p14–15, which spans ~13 Mb (14) .

As for the transfers with the entire chromosome 10, the presence of the transferred derivative chromosomes was confirmed by FISH (Figs. 1Citation and 2Citation ) and microsatellite PCR (Fig. 5)Citation . Contamination of hybrids with mouse DNA could be ruled out in most cases by PCR specific for mouse repetitive DNA elements (data not shown). The sensitivity of this assay was <1 mouse genome equivalent in a background of 1000 human cells.



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  		Fig. 5. Microsatellite PCR analysis of HPKII x 17.I-3 hybrids using D10S1237-specific primers. Arrows, deleted regions in the transferred chromosome in hybrids 32.I-1 and 32.III-3.

 
Allele Loss in the Region of 10p14–p15 in Cervical Cancers.
To determine whether functional loss within the chromosomal region 10p14–15 also contributes to cervical carcinogenesis, LOH analyses were performed for metaplasia (n = 20), CIN1 (n = 5), CIN2/3 (n = 24), and cervical cancers (n = 31). In all cases only histologically verified microdissected areas consisting of 103-104 dysplastic or tumor cells were used. Normal DNA was derived either from blood or from adjacent stromal tissue. Only allelic ratios in the range of 0.0–0.5 were scored as positive and were confirmed in repeat experiments (Fig. 6)Citation . LOH was not detected in any of the metaplasia. Three of 24 (12.5%) CIN2/3 displayed LOH at locus D10S1426, which maps to 10p12.2–p12.3. This locus was also deleted in 3 of 20 (15%) of cervical cancers. Allele loss was also detected in 1 of 5 CIN1 at locus D10S1225, which maps to 10q21.2–q21.3. None of the precancers but 12 of 31 (38.7%) cancers showed LOH for one or more polymorphic marker(s) specific for 10p14–15. The highest frequencies were observed for the loci D10S602 (27%) and D10S501 (28.6%). The frequency of LOH in other regions of chromosome 10 ranged from 10 to 15% for all cervical cancers (Fig. 6)Citation .



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  		Fig. 6. Frequency of allelic losses at defined polymorphic sites of chromosome 10 in 31 cervical carcinomas.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To date, Microcell fusion experiments have identified several chromosomes involved in replicative senescence. Loci with putative senescence genes were localized on chromosome 1 (15) , chromosome 2 (16) , chromosome 3 (17) , chromosome 4 (18) , chromosome 6 (19) , chromosome 7 (20) , chromosome 11 (21) , chromosome 18 (22) , and chromosome X (23) . Of these, only the loss of chromosomes 2 and 4 are linked to the immortal phenotype of HPV-positive cells. Our data suggest that chromosome 10 represents one more candidate chromosome that harbors senescence gene(s), the inactivation of which may contribute to HPV-induced immortalization.

Introduction of a normal copy of chromosome 4 into HeLa cells results in a high number of senescent hybrids (18 , 24) . By somatic cell fusion, it was shown that HeLa and HPKII cells belong to the same complementation group for replicative senescence (25) . It may therefore be argued that HeLa and HPKII cells share the same defects in molecular pathways responsible for senescence. However, the present study provides evidence that the transfer of chromosome 10 can reproducibly induce senescence in HPKII cells. This apparent contradiction that senescence is induced in HPKII cells by complementing gene defects on chromosome 10 can be explained by the model proposed by Sasaki et al. (22) . They suggest that multiple pathways need to be functionally inactivated to acquire an immortal phenotype. However, to restore one of these pathways is sufficient to induce senescence. Of interest, the penetrance by which chromosome 10 can induce the senescent phenotype in HPKII cells is only between 31 and 35%. A similar low percentage of senescent clones has also been described by Sasaki et al. (22) after transfer of chromosome 18 into HHUA endometrial carcinoma cells. One likely explanation for this observation is that a considerable number of hybrids escape senescence because of deletion of parts of the transferred chromosome during clonal expansion of the cells, as confirmed by microsatellite PCR (data not shown). This is not surprising because the hybrids are exposed to strong selective pressure for continued growth, which again may result in the inactivation of this particular senescence gene locus.

We detected a high frequency (38.7%) of LOH at 10p14–p15 in cervical cancers. These findings underline the significance of our functional data, thereby suggesting that inactivation of genes within this region of chromosome 10 may represent an essential step in cervical carcinogenesis. Notably, none of the precancers showed significant LOH (allelic ratios <0.5) for any of the polymorphic markers on 10p14–p15. This may suggest that functional inactivation of putative senescence genes on chromosome 10 is an event that occurs at a late stage in CIN3, possibly even after invasion, and could be a prerequisite for further clonal expansion. Indeed, 2 of 3 cervical carcinomas of tumor stage T1a and 6 of 11 cervical carcinoma of tumor stage T1b, which represent early stage tumors confined to the cervix uteri, showed LOH within 10p14–p15. Several recent studies provide evidence for the presence of tumor suppressor genes on chromosome 10p15. Nishimoto et al. (26) have mapped a 2.7-cM region on 10p15.1, which is required for reactivation of telomerase activity in a human hepatocellular carcinoma cell line. Also by functional analyses, the chromosomal locus responsible for the tumorigenic phenotype of a human prostate cancer cell line was narrowed down to a 1.2-Mb fragment on 10p15.1 (27) .

Because none of the 15 genes thus far mapped to chromosome 10p14–p15 represent obvious candidates for tumor suppressor genes (14) , we will continue to functionally dissect this region further. In this context, the bacterial artificial chromosome clone contig of human chromosome 10p15, which spans a region that is frequently deleted in glioma, will be useful (28) .


   ACKNOWLEDGMENTS
 
We thank Dr. Rosemarie Kühne-Heid for histological diagnosis of biopsy material and Dr. Ingrid Hoyer for help in statistical evaluation.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the Deutsche Forschungsgemeinschaft Grant Schn 294/6-1. Back

2 To whom requests for reprints should be addressed, at Gynäkologische Molekularbiologie, Abteilung Frauenheilkunde der FSU Jena, Bachstrasse 18, 07743 Jena, Germany. Phone: 49-3641-933720; Fax: 49-3641-934272; E-mail: matthias.duerst{at}med.uni-jena.de Back

3 The abbreviations used are: HPV, human papillomavirus; LOH, loss of heterozygosity; FISH, fluorescence in situ hybridization; HPK, HPV-immortalized keratinocytes; CIN, cervical intraepithelial neoplasia; pd, population doubling(s). Back

Received 5/29/01. Accepted 8/16/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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