p53 promotes the fidelity of DNA end-joining activity by, in part, enhancing the expression of heterogeneous nuclear ribonucleoprotein G.

Many studies have suggested the involvement of wild-type (wt) p53 in the repair of DNA double-strand breaks (DSBs) via DNA end-joining (EJ) process. To investigate this possibility, we compared the capacity and fidelity of DNA EJ in RKO cells containing wt p53 and RKO cells containing no p53 (RKO cells with p53 knockdown). The p53 knockdown cells showed lower fidelity of DNA EJ compared to the control RKO cells. The DNA end-protection assay revealed the association of a protein complex including heterogeneous nuclear ribonucleoprotein G (hnRNP G) with the DNA ends in RKO cells containing wt p53, but not with the DNA ends in RKO cells with p53 knockdown. Depletion of endogenous hnRNP G notably diminished the fidelity of EJ in RKO cells expressing wt p53. Moreover, an ectopic expression of hnRNP G significantly enhanced the fidelity of DNA EJ and the protection of DNA ends in human cancer cells lacking hnRNP G protein or containing mutant hnRNP G. Finally, using recombinant hnRNP G proteins, we demonstrated the hnRNP G protein is able to bind to and protect DNA ends from degradation of nucleases. Our results suggest that wt p53 modulates DNA DSB repair by, in part, inducing hnRNP G, and the ability of hnRNP G to bind and protect DNA ends may contribute its ability to promote the fidelity of DNA EJ.

Bmi-1 cooperates with human papillomavirus type 16 E6 to immortalize normal human oral keratinocytes.

Bmi-1 is a member of the polycomb group (PcG) transcription repressors and is implicated in human carcinogenesis. In normal human oral keratinocytes (NHOK), we found that exogenous Bmi-1 expression significantly extended the replicative life span without causing cellular immortalization. Immortalization of NHOK occurs only in combination with human papillomavirus type 16 E6 (HPV-16 E6) but not with E7. During immortalization of NHOK by sequential expression of exogenous Bmi-1 and E6, telomerase activation was observed only after the cells had overcome crisis. Genetic analysis with E6 deletion mutants revealed that the intact second zinc finger domain (amino acids 118-122) was necessary for its cooperative effects with Bmi-1 in the immortalization process. Using these mutants, we found that the increased telomerase activity was closely associated with cell immortalization by Bmi-1 and E6, whereas p53 degradation was not. Using microarray analysis, we identified genes that are immortalization-specific and may participate in the process of NHOK immortalization by Bmi-1 and HPV-16 E6. Our results provide new information on the roles of Bmi-1 and HPV-16 E6 in the multi-step process of oral epithelial carcinogenesis.

Abnormal DNA end-joining activity in human head and neck cancer.

In human cells, DNA double-strand breaks (DSBs) are repaired primarily by the DNA end-joining (EJ) process and thus, abnormal DNA EJ activities lead to an accumulation of mutations and/or aneuploidy, resulting in genetic instability of cells. Since genetic instability is the hallmark of cancer cells, we studied the DNA EJ activities of normal, non-malignant immortalized and malignant human epithelial cells to investigate the association between DNA EJ and carcinogenesis. We found a significant diminution of precise (error-free) DNA EJ activities in non-malignant immortalized human oral keratinocytes (HOK-16B) and human head and neck squamous cell carcinoma (HNSCC) cells compared to that in normal human oral keratinocytes (NHOK). Moreover, abnormal DNA EJ activities were detected exclusively in HOK-16B and HNSCC cells due to microhomology-mediated and non-microhomology-mediated end-joining activities in these cells. These data indicated that aberrant DNA EJ activity may be partly responsible for genetic instability and oncogenic transformation.

HPV-16 E6 oncoprotein impairs the fidelity of DNA end-joining via p53-dependent and -independent pathways.

We previously reported that the E6 oncoprotein of high-risk human papillomavirus (HPV) caused genetic instability and oncogenesis by disrupting cellular DNA repair. Here, to investigate the effect of different domains of E6 on DNA double-strand break (DSB) repair, we infected normal human oral fibroblasts (NHOF) with retroviruses expressing wild-type (wt) or mutant (mt) HPV-16 E6 and examined the cellular DNA end-joining (EJ) activity. The cells expressing E6 showed not only a diminution of error-free DNA EJ but also an increase in erroneous DNA EJ capacity if compared with cells without wt E6. Analysis of DNA EJ activities from the cells expressing mt HPV-16 E6 indicated that binding to p53 and the presence of both intact zinc finger domains of E6 are necessary for inducing the E6-mediated aberrant DNA EJ activity. Also, deletion of the PDZ binding C-terminal region reduced this activity by 50%. These findings suggest that E6 can disrupt the fidelity of DSB repair via both p53-dependent and -independent pathways and the impaired fidelity might contribute to the development of genetic instability found in HPV-associated cancer.

Introduction of human telomerase reverse transcriptase to normal human fibroblasts enhances DNA repair capacity.

PURPOSE: From numerous reports on proteins involved in DNA repair and telomere maintenance that physically associate with human telomerase reverse transcriptase (hTERT), we inferred that hTERT/telomerase might play a role in DNA repair. We investigated this possibility in normal human oral fibroblasts (NHOF) with and without ectopic expression of hTERT/telomerase. EXPERIMENTAL DESIGN: To study the effect of hTERT/telomerase on DNA repair, we examined the mutation frequency rate, host cell reactivation rate, nucleotide excision repair capacity, and DNA end-joining activity of NHOF and NHOF capable of expressing hTERT/telomerase (NHOF-T). NHOF-T was obtained by transfecting NHOF with hTERT plasmid. RESULTS: Compared with parental NHOF and NHOF transfected with empty vector (NHOF-EV), we found that (a) the N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation frequency of an exogenous shuttle vector was reduced in NHOF-T, (b) the host cell reactivation rate of N-methyl-N'-nitro-N-nitrosoguanidine-damaged plasmids was significantly faster in NHOF-T; (c) the nucleotide excision repair of UV-damaged DNA in NHOF-T was faster, and (d) the DNA end-joining capacity in NHOF-T was enhanced. We also found that the above enhanced DNA repair activities in NHOF-T disappeared when the cells lost the capacity to express hTERT/telomerase. CONCLUSIONS: These results indicated that hTERT/telomerase enhances DNA repair activities in NHOF. We hypothesize that hTERT/telomerase accelerates DNA repair by recruiting DNA repair proteins to the damaged DNA sites.

Senescence occurs with hTERT repression and limited telomere shortening in human oral keratinocytes cultured with feeder cells.

We investigated the phenotypic and molecular alterations in normal human oral keratinocytes (NHOK) during in vitro replication in two different culture conditions. The cells were cultured either in chemically defined Keratinocyte Growth Medium (KGM) without feeder layers or in serum-containing flavin-adenine dinucleotide (FAD) medium with feeder layers. Primary NHOK underwent 22 +/- 3 population doublings (PDs) in KGM and 42 +/- 4 PDs in FAD medium, reflecting 52% increase in replication capacity with feeder layers. In both culture conditions, exponentially replicating NHOK demonstrated telomerase activity and expression of human telomerase reverse transcriptase (hTERT) gene. Telomerase activity and hTERT expression were rapidly diminished in senescing NHOK, which exhibited small decrease of telomere length for the remaining limited cellular replications until the complete arrest of cell division. However, telomere length in senescent NHOK was 6.7 +/- 0.5 kilobase pairs (kbps), significantly longer than that (5.12 kbps) of senescent human fibroblasts. The onset of senescence was accompanied with marked induction of p16(INK4A), and this occurred in both culture systems using either KGM or FAD medium. These results indicate that replicative senescence of NHOK is associated with loss of telomerase activity followed by limited telomere shortening.

Telomere shortening does not occur during postmaturational aging in situ in normal human oral fibroblasts.

We investigated whether the telomere length, i.e. mean terminal restriction fragment (TRF) length, decreases during in situ aging in normal human oral fibroblasts (NHOF). For this purpose, NHOF cultures were established from 50 different donors and tested after 14 population doublings (PD) when the cells were replicating exponentially. Telomere-specific Southern blotting and digital quantitation showed that the mean (+/-standard error (S.E.)) TRF length of all tested cultures was 7.72+/-0.17 kbps. The plot of TRF mean length versus donor age showed high variability in individual length with an apparent average decline of -7.8 bp per year of age, which was not statistically significant (r=0.11; P>0.1). These data indicate that telomere shortening does not occur during donor aging in situ, and therefore, is not physiologically relevant for NHOF.

Senescence-associated genes in normal human oral keratinocytes.

The current study was undertaken to identify senescence-associated (SA) genes in cultured normal human oral keratinocytes (NHOK). Primary NHOK were serially subcultured in vitro as dispersed cells in low (0.15 mM) Ca(2+) medium until senescence. The SA genes of NHOK were identified by comparing the expression levels of 3195 human genes between exponentially replicating and senescing cultures. Approximately 5% of the screened genes were upregulated in senescing NHOK by a factor greater than 3 compared with rapidly dividing NHOK culture. Among them, we identified discrete gene groups, i.e., cyclin-dependent kinase inhibitors, G-protein-coupled receptors, apolipoproteins, matrix metalloproteinases, and mitochondrial proteins. To validate the microarray results, we confirmed the enhanced expression of a few selected SA genes, i.e., gpr1, apo-D, apo-E, apo-L, mmp-1, mmp-3, cyb561, cyp1b1, and cyp4b1, by reverse transcription-PCR. These SA genes were upregulated in three independent cultures of NHOK at high population doubling (PD) levels compared with those of low PDs. The enhanced expression of these SA genes was also found in senescing NHOK maintained in 3T3 feeder cell system, as well as in the chemically defined medium containing low Ca(2+). These results indicate that the onset of senescence in NHOK is associated with altered expression of the SA genes, which represent discrete gene groups, independently of the donor variation or culture conditions.

The telomeric length and heterogeneity decrease with age in normal human oral keratinocytes.

We have examined the telomere length in NHOK explanted from 28 donors between the ages of 21 and 84 years. Genomic DNA was isolated from exponentially replicating NHOK and digested with HinFI to yield terminal restriction fragments (TRF). The TRF length ranged from 4.1 to 7.0 kbp with a mean of 5.3 +/- 0.8 kbp, which was significantly shorter than that (8.9 +/- 1.0 kbp) of normal human oral fibroblasts (NHOF). The TRF length was inversely correlated to the increase of donor age in NHOK (m=-23 bp per year; r=-0.60; P<0.001). Also, the heterogeneity of TRF length in cultured NHOK decreased with increased donor age (r=-0.38, P<0.05). These data indicated that clonogenic NHOK cells had replicated in situ and showed a progressive shortening of TRF length. The short telomere length and decreased telomeric length heterogeneity in immortalized cells suggested that there is a critical minimum for cell survival.