Telomere erosion triggers growth arrest but not cell death in human cancer cells retaining wild-type p53: implications for antitelomerase therapy

Telomerase activity in tumours is often associated with p53 mutation. Many antitelomerase therapies take advantage of the inability of cells expressing mutant p53 to undergo replicative senescence, since this allows telomere erosion to continue until 'crisis', hence providing the desired cytotoxic effect. However, some tumour types, including breast, melanomas and thyroid, retain wild-type p53 function and the effectiveness of antitelomerase therapies in such tumour cells have not been adequately addressed. To explore this, we made use of two thyroid cancer cell lines K1 and K2, which retain wt p53. Telomere erosion induced by the expression of a dominant-negative (DN) hTERT resulted in delayed onset of growth arrest in K1 and K2 cells, reminiscent of replicative senescence, with low levels of BrdU labelling and apoptosis, associated with high p21(WAF1) and senescence-associated beta galactosidase expression. In contrast, abrogation of p53 function by the expression of HPV16 E6 in K1 and K2 cells either at the same time as DNhTERT or just prior to the onset of senescence allowed cells to continue growing until 'crisis'. Likewise, microinjection of a p53 neutralizing antibody into 'senescent' K1 DNhTERT cells permitted re-entry into the cell cycle. We conclude that thyroid tumour cells with wild-type p53 retain an intact p53-mediated growth arrest response to telomere erosion. This raises the intriguing question of why, therefore, p53 mutation is not selected for in such cancers, and also calls into question the therapeutic value of telomerase inhibitors in such cases.     

Human wild-type p53 adopts a unique conformational and phosphorylation state in vivo during growth arrest of glioblastoma cells.

The wild-type (wt) human tumor-suppressor gene product, p53, and its mutant form have been analysed in an in vivo system in which the inducible expression of wt p53 results in growth arrest in the G1 phase of the cell cycle. Two major pools of p53 are detected after wt p53 expression by their differential reactivity with the p53 monoclonal antibodies PAb 421 and 1801 as well as the mutant and wt-specific monoclonal antibodies PAb 240 and 1620; one pool contains wt and mutant p53 and is characterized as having a mutant conformation, whereas the other pool contains only wt p53 with a wt conformation. As G1 arrest is entered, the amount of wt p53 associated with the mutant pool decreases, such that by 12 h free wt and mutant p53 are the major pools. Two-dimensional gel analysis of the p53 pools revealed that free wt p53 is phosphorylated to a greater degree than mutant p53, which correlated with the loss of the PAb 421 epitope on wt p53. In summary, the ability of wt p53 to exert an antiproliferative effect correlates with the presence of a unique conformational state of wt p53 characterized by increased phosphorylation and the loss of both the PAb 421 epitope and association with mutant p53 pool, whereas mutant p53 is unable to assume this conformational state.     

Adenovirus-mediated transfer of wild-type p53 gene results in apoptosis or growth arrest in human cultured gastric carcinoma cells.

We examined the susceptibility of six human gastric carcinoma cell lines to infection with recombinant p53 adenovirus vector (AxCA-p53). AxCA-p53 infection at a muliplicity of infection (MOI) of 50 resulted in apoptotic cell death (MKN-1 cells), growth arrest (MKN-45, MKN-74 and KATO-III cells), or non-effectiveness (TMK-1 and OCUM-2M cells). Western blot analysis revealed increasing expression levels of p21/WAF1 protein after infection with AxCA-p53 in all the cell lines. After infection with AxCA-p53, the expression levels of bax or bcl-XL protein changed in MKN-1, but not in the other cell lines. These results suggest that the apoptotic pathway (dependence on the expressions of bcl-2 family proteins) dominates the growth arrest pathway (dependence on the expressions of p21/WAF1 protein) after infection with AxCA-p53. Thus, the bcl-2 family might play a crucial role in p53-mediated growth arrest and apoptosis in human gastric carcinoma cells.     

Expression of wild-type p53 in human A673 cells suppresses tumorigenicity but not growth rate

The p53 gene has been found to be mutated in many different kinds of human cancers. In a previous study, expression of exogenous wild-type p53 in human osteosarcoma cells by retrovirus-mediated gene transfer resulted in marked enlargement of cell size, reduced growth rate in culture and loss of tumorigenicity in nude mice. Here we examine the effects of expression of wild-type or mutated p53 on human peripheral neuroepithelioma (PNET) A673 cells; these cells contained apparently normal alleles of the p53 gene but did not express a detectable quantity of p53 protein. Various characteristics of the p53-expressing cells were examined including morphology, growth rate, soft-agar colony formation, and tumorigenicity in nude mice. In contrast to osteosarcoma Saos-2 cells, expression of wild-type or mutant p53 protein in A673 cells had no effect on morphology or growth characteristics. However, clones expressing wild-type p53 protein had reduced ability to form colonies in soft agar and tumors in nude mice. To substantiate the genotype of wild-type p53-expressing cells, the proviral p53-encoding DNA of one cell clone was amplified by the polymerase chain reaction and sequenced. We concluded that expression of a single allele of the wild-type p53 gene was sufficient to suppress PNET A673 tumorigenicity but had no detectable effect on growth rate in culture.     

Cisplatin-induced p53-independent growth arrest and cell death in cancer cells

Classical Kaposi's sarcoma (CKS) is a rare indolent proliferative disease which is particularly prevalent among Jews of Ashkenazi and Mediterranean origin. To define guidelines for its comprehensive management, we conducted a retrospective analysis of 123 patients, focusing mainly on treatment modalities. The CKS-related mortality was 4% (5 patients). Of the 39 patients for whom observation only was the primary approach, 15 (38%) remained progression-free for 1-83 months (median, 4 months). Twenty-nine of the 52 (56%) patients who underwent surgery as the primary approach remained recurrence-free for 1-162 months (median, 15 months). Radiotherapy achieved an objective response in 74 courses (85%), including 50 (58%) complete responses. Symptomatic relief was reported in 95% of the patients. Vinblastine (27 series) achieved an objective response in 73% of series, including 22% complete responses. Multivariate analysis of time to progression with observation alone identified immunosuppression as the only significant independent factor that predicted disease progression. Our study suggests that observation alone may be sufficient for immunocompetent asymptomatic patients; symptomatic resectable lesions are suitable for simple excision; and more advanced disease or unresectable lesions require radiotherapy. If disease is extensive or the other approaches fail, chemotherapy is appropriate. Tailoring the treatment for CKS is an integrative process, requiring good understanding of the role of each available modality in the different clinical disease settings.     

Retinoblastoma protein-initiated cellular growth arrest overcomes the ability of cotransfected wild-type p53 to induce apoptosis

The retinoblastoma gene, RB, participates in the regulation of the G1/S-phase transition and in p53-mediated apoptosis. We have previously reported that stably transfected RB functions as a growth and tumour suppressor in HTB9 human bladder carcinoma cells, which carry a mutation of the p53 gene at codon 280 and lack RB expression. To elucidate the potential role of RB in the regulation of p53-mediated apoptosis, we transfected a wt p53 expression plasmid under the control of the human cytomegalovirus promoter into parental and RB-transfected HTB9 cells. The p53(+)/RB(-)cells were susceptible to apoptosis under various experimental conditions: 1) incubation in serum-free culture for 72 h, 2) short-term (6 h) or long-term (48 h) exposure to etoposide, and 3) culturing in soft agar. In contrast, p53(+)/RB(+)cells were significantly resistant to apoptosis under similar conditions and exhibited efficient growth arrest, as measured by laser scanning cytometry. Tumorigenicity in nude mice of parental HTB9 cells was lost by exogenous expression of wt p53. Likewise, none of mice injected subcutaneously with either p53(-)/RB(+)or p53(+)/RB(+)cells developed tumours, indicating that RB allows suppression of tumorigenesis, regardless of p53 status. These results suggest that the growth-inhibitory function of RB may overcome the ability of wt p53 to induce apoptosis.     

Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells

The present study assessed the role of adenoviral vector-mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines (n=5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53-transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-beta-Gal-transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols.     

p53 and Egr-1 additively suppress transformed growth in HT1080 cells but Egr-1 counteracts p53-dependent apoptosis.

The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFbeta1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFbeta1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.     

Mutant p53 can provoke apoptosis in p53-deficient Hep3B cells with delayed kinetics relative to wild-type p53

Wild-type (wt) p53 frequently induces apoptosis when expressed in tumor cells whereas mutant p53 acts as an oncoprotein and consequently, stimulates cell proliferation. We report here exceptions to that rule. p53 conformational mutant 175H and DNA contact mutant 273H provoke apoptosis in human p53-deficient Hep3B hepatoma cells with delayed kinetics relative to wt p53. Similarly, c-Myc strongly stimulates apoptosis in these cells. In contrast, viral oncoproteins E1A and E7, and the cellular oncoprotein MDM-2, fail to elicit cytocidal responses. Efficient apoptotic cell death by mutant p53 requires oligomerization as 175H and 273H with deletions between amino acid residues 326 and 347 of the oligomerization domain are nontoxic. Apoptosis by mutant or wt p53 was significantly inhibited by the serine protease inhibitor AEBSF but not by the inactive analog AEBSA. Together, these results suggest that a wt p53-independent control mechanism is operational in Hep3B cells that eliminates cells upon sensing illegitimate proliferation signals originating from certain oncoproteins, including mutant p53 and Myc. We suggest that some tumor cell types lack p53 altogether because they tolerate neither wild-type nor mutant forms of the protein.     

Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway

Ling Zhi-8 (LZ-8), an immunomodulatory protein, is derived from and has been cloned from the medicinal mushroom Ganoderma lucidum (Reishi or Ling Zhi); this protein exhibits immunomodulating and antitumor properties. We investigated the effects of recombinant LZ-8 protein (rLZ-8) on the proliferation of A549 human lung cancer cells. Here, we showed that rLZ-8 inhibits cell growth and that this is correlated with increased G(1) arrest. The treatment of A549 cells with rLZ-8 activated p53 and p21 expression, and both the G(1) arrest and the antigrowth effect were found to be p53 dependent. It was further demonstrated that rLZ-8 inhibited tumor growth in mice transplanted with Lewis lung carcinoma cells. Interestingly, rLZ-8 treatment was found to lead to nucleolar stress (or ribosomal stress) as evidenced by inhibition of precursor ribosomal RNA synthesis and reduced polysome formation in A549 cells. These changes resulted in an increasing binding of ribosomal protein S7 to MDM2 and a decreased interaction between MDM2 and p53. Taking these results together, we have identified a novel rLZ-8 antitumor function that positively modulates p53 via ribosomal stress and inhibits lung cancer cell growth in vitro and in vivo. Our current results suggest that rLZ-8 may have potential as a therapeutic intervention for the treatment of cancers that contain wild-type p53 and high expression of MDM2.     

Expression of a non-functional p53 affects the sensitivity of cancer cells to gemcitabine.

Gemcitabine is a relatively new agent with promising activity in solid tumors. Few data are available regarding mechanisms of resistance to gemcitabine downstream from the drug-target interaction. The present study was performed to gain insight into the role of p53 status on the cytotoxicity of gemcitabine on cancer cells. Drug sensitivity, drug metabolism, cell kinetics and drug-induced apoptosis were compared in 2 lines derived from the mammary adenocarcinoma MCF-7: the wildtype p53 (wt-p53) containing MN-1 cell line and, the MDD2 line containing a dominant negative variant of the p53 protein (mut-p53). The MDD2 cell line was significantly more resistant to gemcitabine cytotoxicity than the MN-1 cell line. The resistant phenotype could not be attributed to a defective gemcitabine activation/degradation pathway or altered levels of expression of intracellular targets. Although both cell lines exhibited p53 accumulation, MN-1 but not MDD2 cells, displayed p21(WAF1) induction after exposure to gemcitabine. Gemcitabine induced an S-phase arrest in both cell lines. A more pronounced block in G1 phase, however, was observed in MN1 cells. Exposure to gemcitabine induced a higher degree of apoptosis in MN-1 than in MDD2 cells. This corresponded with suppression of Bcl-2 and Bcl-X/L expression in wt-p53 cells exposed to gemcitabine whereas Bcl-2 levels remained stable and Bcl-X/L levels increased in mut-p53 cells exposed to gemcitabine. We conclude that the p53 status of cancer cells influences their sensitivity to gemcitabine cytotoxicity. Our evidence suggests that loss of p53 function leads to loss of cell cycle control and alterations in the apoptotic cascade, conferring resistance to gemcitabine in cancer cell lines displaying a mut-p53.     

Therapeutic potential of recombinant p53 overexpression in breast cancer cells expressing endogenous wild-type p53

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21^{WAF 1/CIP1} protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.