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Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16^INK4A/pRb¹

NIINA VEITONMÄKI^2,, JONAS FUXE^2,, MAGNUS HULTDIN, GÖRAN ROOS, RALF F. PETTERSSON and YIHAI CAO^,3

Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden;
Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, S-171 77 Stockholm, Sweden; and
Department of Medical Biosciencies, Pathology, Umeå University, 90187 Umeå, Sweden

³Correspondence: Microbiology and Tumour Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail: yihai.cao{at}mtc.ki.se

SPECIFIC AIMS

We investigated 1) whether human TERT could immortalize bovine capillary endothelial (BCE) cells; 2) whether immortalized BCE cells retain the same original endothelial cell features as primary BCE cells; and 3) the molecular mechanisms of cellular immortalization by hTERT in these heterologous bovine primary endothelial cells.

PRINCIPAL FINDINGS

1. hTERT-immortalized BCE cells retain the same endothelial features as primary BCE cells
Primary BCE cells cultured in vitro under optimal conditions enter senescence after 40–45 PDLs. Like human cells, they acquire specific senescent characteristics, including positive staining for senescence-associated ß-galactosidase (SA-ß-Gal) and large flattened cell sizes. Stable transfection of hTERT alone led to immortalization of these cells. At the time of submitting this report, the hTERT⁺-BCE cells have passed > 300 PDLs and continue to proliferate as primary young BCE cells, without evidence of obvious altered cell morphology or growth rate. Like telomerase-immortalized human cells, hTERT⁺-BCE cells were unable to form tumors when implanted into immunodeficient SCID mice. These data demonstrate that hTERT alone is able to immortalize bovine endothelial cells without cellular transformation. Moreover, expression of hTERT in BCE cells did not alter their ability to take up acetylated-LDL, an endothelial cell marker. Cell proliferation and migration assays showed that hTERT⁺-BCE cells responded to angiogenesis factors and inhibitors in a similar fashion as primary BCE cells.

2. Immortalization of BCE cells by hTERT is not associated with the net increase of telomere lengths
High telomerase activity was detected in the hTERT⁺-BCE cells, and was maintained for > 200 PDLs (Fig. 1 B, lanes 2–5). In contrast, undetectable levels of telomerase activity were found in senescent BCE cells (Fig. 1B , lane 1) and in presenescent BCE cells (Fig. 1A , lane 1). Surprisingly, despite high telomerase activity, the peak telomere lengths in hTERT⁺-BCE cells were found to shorten during progressive cell doublings (Fig. 1C, D ). At an early PDL of 50, the peak telomere length was similar to the length detected in senescent primary BCE cells, with telomere sizes of 11–12 kb. In later population doublings, they were found to be significantly shorter, ranging from 8.6 to 7.8 in PDL 150 and PDL 200, respectively. The mean telomere lengths in the hTERT⁺-BCE cells at PDLs 150 and 200 were 1 kb shorter than those seen in senescent BCE cells (Fig. 1D , lower panel). In higher PDLs the telomere lengths seemed to be stabilized at this size (data not shown).

View larger version (67K): [in this window] [in a new window]   Figure 1. Telomerase activity and telomere length (TRF). A) Telomerase activity in primary BCE cells at PDL 30 was measured. TRF8, a control template (lane 3), served as a positive control. B) Telomerase activity in senescent BCE cells at PDL 35 (lane 1) and hTERT+-BCE cells at PDLs between 50 and 200 (lanes 2–5). C) Telomere lengths of senescent primary BCE cells (lane 1) and hTERT+-BCE cells at various PDLs were detected by Southern blot analysis. D) Presentation of the peak (upper panel) and mean (lower panel) telomere lengths in primary BCE cells (lane 1) and hTERT+-BCE cells at various PDLs (lanes 2–5).

3. Repression of p16 and p21 in hTERT⁺-BCE cells
The finding that the peak TRFs continued to decrease in the telomerase expressing cells suggested that BCE immortalization might be mediated by alternative mechanisms rather than lengthening of telomeres. We found that pRb was hyperphosphorylated in the hTERT⁺-BCE cells as compared with primary BCE cells. Note levels of p16 and p21 accumulated in senescent BCE cells (Fig. 2 A, lane 2) compared with BCE cells at lower PDLs (Fig. 2A , lane 1). In hTERT⁺-BCE cells at both early and late number of PDLs, expression of p16 and p21 (Fig. 2A , lanes 3–5) was repressed to levels detected in nonsenescent young BCE cells, or even less. Expression of p53 remained at similar levels in both senescent BCE and hTERT⁺-BCE cells (Fig. 2A ).

View larger version (63K): [in this window] [in a new window]   Figure 2. A) Status of p16 and p21 in primary BCE and hTERT+-BCE cells. Expression of the proteins p16, p21, p53, and CDK4 was studied in primary BCE cells at 25 (lane 1) and 35 (lane 2) PDLs and in hTERT+-BCE cells at various PDLs (lanes 3–5). Expression of calnexin was used as standard quantitation of equal loading of protein samples in each lane. B–F) Reinduction of a senescent-like phenotype by p16.hTERT+-BCE cells incubated with the demethylation agent 5-azadeoxycytidine (Aza) stained for the SA-ß-gal marker (B) and for expression of p16 and p21 (F, lane 2). The expression of p16 was also analyzed by immunofluorescence (C) and immunoblotting (F, lane 3) after infection of hTERT+-BCE cells with an adenovirus encoding the p16 cDNA (Adp16). 7 days after infection, the cells were stained with the SA-ß-gal marker (E). A control virus expressing the green fluorescent protein (AdGFP) was used as a negative control (D). G) Expression of the DNA methyltransferase DNMT1 in presenescent BCE cells at PDL 30 (lane 1), senescent BCE cells at PDL 35 (lane 2), hTERT+-BCE cells at various PDLs (lanes 3–5), and after treatment with Aza (lane 6).

4. Reactivation of p16, p21, and a senescence-like phenotype by demethylation
To determine whether promoter methylation could be involved in inactivation of p16 in hTERT⁺-BCE cells, we treated the cells with the demethylating agent 5-azadeoxycytidine (Aza). This treatment resulted in reinduction of a senescence-like phenotype, with larger flattened cells that exhibited SA-ß-gal activity (Fig. 2B ) and re-expression of both p16 and p21 (Fig. 2F , lane 2) compared with untreated cells (Fig. 2F , lane 1). This effect of 5-azadeoxycytidine suggested that telomerase expression is not sufficient to maintain the immortalized phenotype without the concomitant inactivation of p16.

5. Reinduction of a senescence-like phenotype by ectopic expression of p16
If inactivation of p16 was critical for hTERT-mediated immortalization and bypass of cellular senescence, re-expression of p16 alone should mimic the effects seen by treatment with 5-azadeoxycytidine and reverse the immortalized phenotype of the hTERT⁺-BCE cells. To test this hypothesis, we transduced hTERT⁺-BCE cells with an adenovirus encoding p16 (Adp16). Expression of p16 in the hTERT⁺-BCE cells was confirmed by immunofluorescence (Fig. 2C ) and immunoblotting (Fig. 2F , lane 3), resulting in reinduction of a senescent-like phenotype (Fig. 2E ) and growth inhibition.

6. Elevation of DNMT1 expression levels in hTERT⁺-BCE cells
The activity of DNMT1, the prototypic mammalian DNA methyltransferase, has previously been reported to correlate with inactivation of both p16 and p21. We analyzed the expression level of DNMT1 in the immortalized cells. The results showed that the DNMT1 levels were 5- to 7-fold higher in all PDLs of hTERT⁺-BCE cells (Fig. 2G , lanes 3–5) as compared with senescent BCE cells (Fig. 2G , lane 2), and 2- to 2.5-fold higher compared with presenescent BCE cells (Fig. 2G , lane 1). Treatment with 5-azadeoxycytidine completely abolished DNMT1 expression in the hTERT⁺-BCE cells (Fig. 2G , lane 6).

CONCLUSIONS AND SIGNIFICANCE

Our data demonstrate that human TERT alone is sufficient to immortalize primary bovine capillary endothelial cells and that immortalization is mediated through inactivation of crucial cellular senescent machineries, including p16 and pRb (Fig. 3 ). Even though stable and high levels of telomerase activity are detected in various PDLs, telomere lengths are significantly shortened in hTERT-immortalized BCE cells compared with senescent BCE cells. This is surprising because the key function of hTERT is thought to be to maintain and extend the telomeric repeats at the ends of chromosomes, which are essential for genomic integrity and stability. In addition to our findings, several independent studies using primary human cells have reported similar results. Two models have been put forward to explain this phenomenon. One suggests that telomerase lengthens the shortest telomeres; the other proposes that telomerase promotes cell proliferation and prevents telomeric fusions. Although these hypotheses may explain the role of telomerase in maintenance of the shortest telomere lengths in the immortalized cells, they cannot explain how telomerase enforces cells to bypass cellular senescence without net lengthening of telomeres. Our data suggest that subsequent repression of cellular senescent machineries by hTERT activity might play an important role in cellular immortalization.

View larger version (21K): [in this window] [in a new window]   Figure 3. Stable transfection of human TERT alone was sufficient to immortalize primary BCE cells. hTERT-immortalized BCE cells displayed the same biological features as primary young BCE cells and were incapable of forming tumors in immunodeficient mice. The telomere lengths in these cells were progressively shortened as they bypassed senescence, despite high telomerase activity. Analyses of crucial cellular senescent machinery revealed that the expression of both p16 and p21 was repressed and that pRb was hyperphosphorylated. At least the p16 gene seemed to be inactivated through DNA methylation; in agreement with this, expression levels of the prototypic DNA methyltransferase DNMT1 were elevated in the hTERT-positive cells.

Our experiments showed that at least the p16 gene is inactivated by hypermethylation and that expression of DNMT1 is elevated in the immortalized cells. We therefore suggest dual activities of hTERT in cellular immortalization; apart from its role in stabilizing telomeric ends, hTERT may influence the function of senescence machineries and directly or indirectly be involved in gene inactivation. The fact that the expression level of DNMT1 is increased in hTERT-immortalized cells may not be explained simply by an increased cell proliferation, since we have observed that these expression levels are higher than those detected in young and proliferating primary BCE cells. In addition to DNMT1, it has recently been shown that DNMT3b is involved in DNA methylation and gene silencing in cancer cells and that the activity of DNMT1 and DNMT3b is essential for optimal neoplastic proliferation. It is not known how these DNA methyltransferases coordinate their activities in regulating gene expression. Whether TERT has any direct or indirect effects on their expression levels remains to be studied.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0599fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0599fje

² The first two authors contributed equally to this work.

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