Induction of senescence in human malignant glioma cells by p16INK4A

Abstract

p16INK4A is a G1-speci®c cell cycle inhibitor which maps to human chromosome 9p21, a region frequently mutated or deleted in cancer cell lines and primary tumors. In glioblastomas the frequency of homozygous deletions is 40 ± 70% making it one of the most common mutations in this tumor type. We have analysed the signi®cance of the loss of this gene in gliomas by introducing the cDNA for p16INK4A into the human glioma cell line U-1242 MG which has a deleted CDKN2 locus. We used the tetracycline repressable vector system and obtained two stably transfected clones that expressed p16INK4A upon induction. p16INK4A expression caused a G1 arrest and enlargement of the cells similar to that of senescent cells. When staining for Senescence-Associated b-galactosidase activity, described to be speci®c for senescent cells, we could show that the enlarged cells speci®cally gave a positive staining reaction. This senescence phenotype was dependent on the continuous expression of p16INK4A since it was reversed upon reintroduction of tetracycline suppression. Thus, the induced expression of p16 INK4A in these glioma cells reverted their immortal phenotype and caused an immediate cellular senescence.

Involvement of the Ink4 proteins p16 and p15 in T-lymphocyte senescence

“…However, a reduced expression of CD28 has previously been observed during T-lymphocyte senescence (Perillo et al, 1993), an e ect that was observed also in our system. Furthermore, we observed an induction of SA-b-gal activity in the aged Tlymphocytes, which has been shown to be a biomarker for senescence in other cellular systems (Dimri et al, 1995;Uhrbom et al, 1997). This demonstrates that SAb-gal activity can be used also for analysing senescence in T-lymphocytes.…”

“…At the initiation of the culture the majority of the cells expressed CD28 (Figure 2a), whereas the non-proliferating cells at the end of the culture had virtually abolished their expression of this marker (Figure 2b). b-gal (b-gal) staining at pH 6.0, referred to as Senescence-Associated b-galactosidase (SA-b-gal), has been shown to be a reliable marker for senescence in other cellular systems by (Dimri et al, 1995;Uhrbom et al, 1997). We found that 1 ± 2% of un-stimulated T-cells have detectable SA-b-gal activity.…”

Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2′-deoxycytidine treatment induces a senescence-like state

“…Moreover, upregulation of p16 ink4a a member of the ink4 family, has been observed during the late stages of senescence, where proliferation ceases terminally (Alcorta et al, 1996). Consistent with a role in regulating senescence, p16 ink4a expression was lost in mammary epithelial cells that escaped from the M0 proliferation block (Foster et al, 1998) and ectopic expression of p16 ink4a induced termination of proliferation and a senescence-like state in human glioma cells (Uhrbom et al, 1997).…”

“…In mammary epithelial cells inactivation of p16 ink4a by CpG island methylation was shown to be associated with escape from the M0 proliferation block (Foster et al, 1998). Moreover, engineered expression of p16 ink4a in a human glioma cell line with a p16 ink4a deletion led to the onset of senescence, as determined by induction of senescencespeci®c b-galactosidase activity and was accompanied by changes in cellular morphology (Uhrbom et al, 1997). We observed very similar e ects, including a dramatic increase in cell size in SCC15 cells both in response to re-expression of endogenous p16 ink4a and when p16 ink4a expression was enforced by a retrovirus.…”

The protein kinase C-η isoform induces proliferation in glioblastoma cell lines through an ERK/Elk-1 pathway

“…Both cell lines were isolated from human glioblastomas and various characteristics have been described (Bigner et al, 1981;Bongcam-Rudloff et al, 1991;Uhrbom et al, 1997;Hussaini et al, 1999). U-1242-Z cells are U-1242 cells that stably express PKC-Z (Hussaini et al, 2000).…”