Telomerase suppression by chromosome 6 in a human papillomavirus type 16-immortalized keratinocyte cell line and in a cervical cancer cell line.
BACKGROUND
High-risk human papillomavirus (HPV) types play a major role in the development of cervical cancer in vivo and can induce immortalization of primary human keratinocytes in vitro. Activation of the telomere-lengthening enzyme telomerase constitutes a key event in both processes. Because losses of alleles from chromosome 6 and increased telomerase activity have been observed in high-grade premalignant cervical lesions, we analyzed whether human chromosome 6 harbors a putative telomerase repressor locus that may be involved in HPV-mediated immortalization.


METHODS
Microcell-mediated chromosome transfer was used to introduce chromosomes 6 and 11 to the in vitro generated HPV type 16 (HPV16)-immortalized keratinocyte cell line FK16A and to the in vivo derived HPV16-containing cervical cancer cell line SIHA: Hybrid clones were analyzed for growth characteristics, telomerase activity, human telomerase reverse transcriptase (hTERT) and HPV16 E6 expression, and telomere length. FK16A hybrid clones were also transduced with an hTERT-containing retrovirus to examine the effect of ectopic hTERT expression on growth. Statistical tests were two-sided.


RESULTS
Introduction of human chromosome 6 but not of chromosome 11 to both cell lines yielded hybrid cells that demonstrated crisis-like features (i.e., enlarged and flattened morphology, vacuolation, and multinucleation) and underwent growth arrest after a marked lag period. In the chromosome 6 hybrid clones analyzed, telomerase activity and hTERT messenger RNA (mRNA) expression were statistically significantly reduced compared with those in the chromosome 11 hybrid clones (for telomerase activity, P =.004 for the FK16A hybrids and P =.039 for the SiHa hybrids; for hTERT mRNA expression, P =.003 for the FK16A hybrids). The observed growth arrest was associated with telomeric shortening. Ectopic expression of hTERT in FK16A cells could prevent the telomeric shortening-based growth arrest induced by chromosome 6.


CONCLUSIONS
Chromosome 6 may harbor a repressor of hTERT transcription, the loss of which may be involved in HPV-mediated immortalization.
Steenbergen
R D
RD
Department of Pathology, Unit of Molecular Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Kramer
D
D
Meijer
C J
CJ
Walboomers
J M
JM
Trott
D A
DA
Cuthbert
A P
AP
Newbold
R F
RF
Overkamp
W J
WJ
Zdzienicka
M Z
MZ
Snijders
P J
PJ
D002455:Cell Division; D002461:Cell Line, Transformed; D002880:Chromosomes, Human, Pair 11; D002896:Chromosomes, Human, Pair 6; D004268:DNA-Binding Proteins; D005260:Female; D017930:Genes, Reporter; D006801:Humans; D006822:Hybrid Cells; D015603:Keratinocytes; D018895:Microsatellite Repeats; D027383:Papillomaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012313:RNA; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016615:Telomere / Q000235:genetics / Q000648:ultrastructure; D014162:Transfection; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D004268:DNA-Binding Proteins; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase; D001616:beta-Galactosidase