Functional demonstration of a mortality phenotype associated with 4cen-q23.
Normal human keratinocytes possess a finite replicative lifespan
whereas most advanced squamous cells carcinomas are immortal. The
mechanisms, whose abrogation's can be considered necessary to achieve
immortality, include those involving the negative cell cycle regulators p53,
p161NK4Aa,nd the telomere repair reverse-transcriptase enzyme, telomerase.
Other specific chromosomes have also been demonstrated to carry functions
whose loss is necessary for the development of immortality, through immortal
phenotype reversion upon reintroduction into an immortal cell line.
We demonstrate here the phenotypic reversion of immortal HNSCCderived
keratinocyte cell lines to a mortal growth-arrest upon reintroduction of
a resistance marker tagged wild-type human chromosome 4. We further
demonstrate that this phenotypic reversion occurs only in cell lines, which
display LOH on 4q (BICR6 and BICR31), and not in those with intact
endogenous chromosome 4 copies (BICR3 and BICR19), and that it is
chromosome 4-specific, chromosomes 6, 11, and 15 having no effect on
proliferative lifespan following introduction into BICR6 by MMCT. Through
XMMCT-based truncated chromosomal fragment generation the functional
complementation was localised to 4cen-q23, whilst fine mapping in
segregants arising from the MMCT experiments identified an approximate
1.5Mb locus containing a minimal number of candidate genes.
Through biological assay we have further determined the growth-arrest
to have characteristics of crisis. This was determined through low BrdU
incorporation balanced with high levels of apoptosis to statistically significance
levels «0.05). We found no evidence for involvement of telomeric attrition in
the observed phenotype, through insufficient phenotypic lag (3-10 MPD) and
growth-arrest in the presence of ectopic hTERT expression, suggesting the
operation of an alternative mechanism. This suggests the presence of
gene(s) at 4cen-q23 whose loss is advantageous to the development of
immortality in advanced tumours including HNSCC