Increased radiation-induced apoptosis and altered cell cycle progression of human lung cancer cell lines by antisense oligodeoxynucleotides targeting p53 and p21(WAF1/CIP1)

Lung cancer is difficult to control locally by radiotherapy and is known to have frequently p53 mutations. Previous results have shown that non-small-cell lung cancer (NSCLC) cell lines with nonfunctional p53 have a higher fraction of radiation-induced apoptosis and that apoptosis follows after the release from the G2/M arrest. The aim of the present work was to study whether inhibition of the p53 response in NSCLC cell lines can modulate the G2/M arrest and the induction of apoptosis after ionizing radiation. Antisense oligodeoxynucleotides (As-ODNs) were used to inhibit the p53 response in the cell lines H460 and A549 with functional p53. In addition, H661 with nonfunctional p53 was used. The results have shown that As-ODNs targeting mRNA of p53 and p21 downregulate radiation-induced expression of p53 and p21(WAF1/CIP1). Delayed apoptosis (35.7+/-4.2% in H460, 1.2+/-0.4% in A549 and 72.2+/-6.5% in H661) was observed after cell cycle progression beyond the G2 block, either in the late G2 phase of the same cell cycle being irradiated (H661) or in the G1 phase of the subsequent cell cycle (H460, A549). As-p53 significantly decreased the fraction of G2/M-arrested cells in H460 cells and increased radiation-induced apoptosis at 96 hours by 17.9+/-8.5 and 9.1+/-3.3% to 53.6+/-7.4 and 10.8+/-2.9% in H460 and A549 cells (P<.01), respectively, but had no effect in H661 cells with nonfunctional p53. In addition, As-p21 decreased the fraction of G2-arrested A549 and H460 cells and increased apoptosis by 23.8+/-5.2 and 31.6+/-7.3% to 59.4+/-3.1 and 32.8+/-7.3%, respectively (P<.01). In conclusion, these data show that radiation-induced G2 arrest is decreased in NSCLC cells and radiation-induced apoptosis is increased when p53-responsive pathways are blocked via As-ODN targeting p53 or p21(WAF1/CIP1) mRNA. In view of the fact that p53 and p21 As-ODN had similar effects on radiation-induced apoptosis normalized by their ability to inhibit radiation-induced p21 expression, we concluded that p21 is an important trigger of late ionizing radiation-induced apoptosis.     

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of non-small cell lung cancer cells via the JNK/c-Jun/AP-1 signaling

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of NF-κB in vitro and has afforded some objective responses in individuals with relapsed, refractory MM. However, the activity of PS-341 against non-hematological malignancies remains to be fully elucidated. In this study, we found that PS-341 induced growth arrest and apoptosis of NCI-H520 and -H460 non-small cell lung cancer (NSCLC) cells in conjunction with markedly up-regulated levels of p21^waf1 and p53, and down-regulation of bcl-2 protein in these cells. Also, PS-341 caused phosphorylation of c-Jun NH₂-terminal kinase (JNK) and c-Jun, and enhanced AP-1/DNA binding activities in these cells as measured by western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Interestingly, when the JNK/ c-Jun/AP-1 signal pathway was disrupted by the JNK inhibitor SP600125, the ability of PS-341 to inhibit the growth of NSCLC cells and to up-regulate the levels of p21^waf1 in these cells was blunted, but the expression of p53 was sustained at a high level, suggesting that the JNK/c-Jun/AP-1 signal pathway might mediate the anti-lung cancer effects of PS-341, with p21^waf1 playing the central role. Thus, PS-341 might be useful for the treatment of individuals with NSCLC.     

Cell cycle disturbances and apoptosis induced by topotecan and gemcitabine on human lung cancer cell lines

Apoptosis is a major mode of cell death in response to cytotoxic drug treatment. A correlation between induction of apoptosis and chemosensitivity has been documented in some preclinical models. Topotecan (a topoisomerase I inhibitor) and gemcitabine (a deoxycytidine analogue), two active new drugs for the treatment of lung cancer, were evaluated for their growth inhibitory effect on human lung cancer cell lines and their effect on cell cycle perturbation, apoptosis and apoptosis-related genes. The cytotoxicities of topotecan and gemcitabine on the human lung cancer cell lines H460 (wild-type-p53) and H322 (mutant p53) were determined after 72 h drug exposure employing the MTT assay. The apoptotic index (AI) was assessed by three methods: analysis of morphological changes using May–Grünwald–Giemsa (MGG) staining, the TUNEL assay and FACS analysis. Cell cycle disturbances were studied by FACS and the number of cells expressing p53 and p21 were determined by immunohistochemistry. Both gemcitabine and topotecan had potent growth inhibitory effects in human lung cancer cell lines; combination treatment with these two drugs showed some additivity but no synergism. Induction of apoptosis after treatment was concentration- and time-dependent with both drugs and ic₈₀ concentrations induced the highest values. The DNA histograms at 4, 24, 48 and 72 h indicate that topotecan at ic₈₀ concentrations causes accumulation of cells in S and G2/M phases, whereas gemcitabine at ic₈₀ concentrations causes, accumulation of cells in G1 phase. Both compounds induced p53 and p21 expression in the H460 cell line but not in the H322 cell line; the percentage of cells expressing p53 was highest at ic₈₀ values, whereas the highest percentage of p21 positive cells could be induced with ic₅₀ values. This could suggest that p53 induces cell cycle arrest at low drug concentrations, whereas p53 induces apoptosis at higher concentrations. In conclusion, p53-dependent and independent pathways of apoptosis exist in lung cancer cell lines. Activation of the p53 pathway depends on the induced cellular damage. Understanding the cell cycle disturbances induced by these drugs may help in the design of more rational treatment schedules.     

Novel therapies targeting the myeloma cell and its bone marrow microenvironment.

Novel therapies in multiple myeloma (MM) target not only the tumor cell but also the bone marrow (BM) microenvironment. Thalidomide (Thal), as well as derivative immunomodulatory drugs (IMiDs), directly induce apoptosis or G1 growth arrest in MM cell lines and patient's MM cells which are resistant to melphalan (Mel), doxorubicin (Dox), and dexamethasone (Dex). Although Thal and IMiDs do not alter adhesion of MM cells to bone marrow stromal cells (BMSCs), they inhibit the upregulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion triggered by the binding of MM cells to BMSCs. Proteasome inhibitors represent another potential anticancer therapy targeting the MM cell and the BM microenvironment. The proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis in both human MM cell lines and freshly isolated patient's MM cells which are resistant to Mel, Dox, and Dex. PS-341 inhibits p44/42 mitogen-activated protein kinase (MAPK) growth signaling triggered by IL-6 and induces apoptosis, despite induction of p21 and p27, in p53 wild-type and p53 mutant MM cells. PS-341 adds to the anti-MM activity of dexamethasone and overcomes IL-6-mediated protection against dexamethasone-induced apoptosis. PS-341 blocks the paracrine growth of human MM cells by decreasing their adherence to BMSCs and related NF-kappaB-dependent induction of IL-6 secretion in BMSCs. Moreover, proliferation and MAPK growth signaling of those residual adherent MM cells is also inhibited. Tumor necrosis factor-alpha (TNF-alpha), which is produced by some MM cells, induces only low-level MM proliferation and MAPK activation in MM cells, but markedly upregulates IL-6 secretion from BMSCs and upregulates expression of adhesion molecules (VLA-4 and LFA-1) on MM cells and their receptors (VCAM-1 and ICAM-1) on BMSCs, with resultant increased binding of MM cells to BMSCs. Inhibition of TNF-alpha-induced NF-kappaB activation with PS-341 inhibits both the upregulation of these molecules on MM cells and BMSCs and the resultant increased adhesion. Therefore, inhibiting TNF-alpha and its sequelae may be useful treatment strategies in MM. Our data show that VEGF causes proliferation and enhances migration of MM as well as plasma cell leukemia (PCL) cells. VEGF induced twofold activation of cell migration in MM cells and more than 100-fold activation of cell migration in PCL cells, suggesting an important role of VEGF in the progression of MM to PCL. These data indicate that VEGF plays a pivotal role not only in neoangiogenesis in MM BM but also in proliferation and migration of tumor cells.     

Induction of cell cycle arrest and apoptosis by the proteasome inhibitor PS-341 in Hodgkin disease cell lines is independent of inhibitor of nuclear factor-kappaB mutations or activation of the CD30, CD40, and RANK receptors.

PURPOSE: The malignant Hodgkin and Reed-Sternberg cells of Hodgkin disease (HD) are known to constitutively express high levels of activated nuclear factor kappaB (NF-kappaB), which plays an important role in their survival. The proteasome inhibitor PS-341 has been recently shown to modulate tumor cell proliferation and survival by inhibiting NF-kappaB and modulating critical cellular regulatory proteins, but its activity in cells carrying IkappaBalpha gene mutations has not been reported previously. Experimental Design: The activity of PS-341 in four well-characterized, HD-derived cell lines. Cell proliferation and apoptosis were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) and Annexin-V binding methods, respectively. Cell cycle analysis was determined by flow cytometry. Intracellular protein levels were determined by Western blot. RESULTS: PS-341 demonstrated a strong antiproliferative activity, which was irrespective of the status of mutations in IkappaBalpha and even the presence of CD30, CD40, or RANK receptor activation. This effect was attributable to the induction of apoptosis and cell cycle arrest at the G(2)-M phase. PS-341 not only inhibited nuclear localization of NF-kappaB but also activated the caspase cascade, increased p21 and Bax levels, and decreased Bcl-2 levels. Furthermore, PS-341 enhanced the effect of gemcitabine chemotherapy and potentiated the effect of tumor necrosis factor-related apoptosis-inducing ligand/APO2L and two agonistic antibodies to tumor necrosis factor-related apoptosis-inducing ligand death receptors R1 and R2. CONCLUSIONS: The in vitro activity of PS-341 against HD-derived cell lines suggests that PS-341 may have a therapeutic value for the treatment of HD.     

An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines

The mechanisms of resistance to the antimetabolite gemcitabine in non-small cell lung cancer have not been extensively evaluated. In this study, we report the generation of two gemcitabine-selected non-small cell lung cancer cell lines, H358-G200 and H460-G400. Expression profiling results indicated that there was evidence for changes in the expression of 134 genes in H358-G200 cells compared with its parental line, whereas H460-G400 cells exhibited 233 genes that appeared to be under- or overexpressed compared with H460 cells. However, only the increased expression of ribonucleotide reductase subunit 1 (RRM1), which appeared in both resistant cell lines, met predefined analysis criteria for genes to investigate further. Quantitative PCR analysis demonstrated H358-G200 cells had a greater than 125-fold increase in RRM1 RNA expression. Western blot analysis confirmed high levels of RRM1 protein in this line compared with the gemcitabine-sensitive parent. No significant change in the expression of RRM2 was observed in either cell line, although both gemcitabine-resistant cell lines had an approximate 3-fold increase in p53R2 protein. A partial revertant of H358-G200 cells had reduced levels of RRM1 protein (compared with G200 cells), without observed changes in RRM2 or p53R2. In vitro analyses of ribonucleotide reductase activity demonstrated that despite high levels of RRM1 protein, ribonucleotide reductase activity was not increased in H358-G200 cells when compared with parental cells. The cDNA encoding RRM1 from H358-G200 cells was cloned and sequenced but did not reveal the presence of any mutations. The results from this study indicate that the level of RRM1 may affect gemcitabine response. Furthermore, RRM1 may serve as a biomarker for gemcitabine response.     

The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells

Human multiple myeloma (MM) is a presently incurable hematological malignancy, and novel biologically based therapies are urgently needed. Proteasome inhibitors represent a novel potential anticancer therapy. In this study, we demonstrate that the proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis of human MM cell lines and freshly isolated patient MM cells; inhibits mitogen-activated protein kinase growth signaling in MM cells; induces apoptosis despite induction of p21 and p27 in both p53 wild-type and p53 mutant MM cells; overcomes drug resistance; adds to the anti-MM activity of dexamethasone; and overcomes the resistance to apoptosis in MM cells conferred by interleukin-6. PS-341 also inhibits the paracrine growth of human MM cells by decreasing their adherence to bone marrow stromal cells (BMSCs) and related nuclear factor kappaB-dependent induction of interleukin-6 secretion in BMSCs, as well as inhibiting proliferation and growth signaling of residual adherent MM cells. These data, therefore, demonstrate that PS-341 both acts directly on MM cells and alters cellular interactions and cytokine secretion in the BM millieu to inhibit tumor cell growth, induce apoptosis, and overcome drug resistance. Given the acceptable animal and human toxicity profile of PS-341, these studies provide the framework for clinical evaluation of PS-341 to improve outcome for patients with this universally fatal hematological malignancy.     

Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis

Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation, and apoptosis; hence, perturbed Notch signaling may contribute to tumorigenesis. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in Africa and Asia. The mechanisms that orchestrate the multiple oncogenic insults required for initiation and progression of HCC are not clear. We constitutively overexpressed active Notch1 in human HCC to explore the effects of Notch1 signaling on HCC cell growth and to investigate the underlying molecular mechanisms. We show here that overexpression of Notch1 was able to inhibit the growth of HCC cells in vitro and in vivo. Biochemical analysis revealed the involvement of cell cycle regulated proteins in Notch1-mediated G(0)/G(1) arrest of HCC cells. Compared with green fluorescent protein (GFP) control, transient transfection of Notch1 ICN decreased expression of cyclin A (3.5-fold), cyclin D1 (2-fold), cyclin E (4.5-fold), CDK2 (2.8-fold), and the phosphorylated form of retinoblastoma protein (3-fold). Up-regulation of p21(waf/cip1) protein expression was observed in SMMC7721-ICN cells stably expressing active Notch1 but not in SMMC7721-GFP cells, which only express GFP. Furthermore, a 12-fold increase in p53 expression and an increase (4.8-fold) in Jun-NH(2)-terminal kinase activation were induced in SMMC7721-ICN cells compared with SMMC7721-GFP cells. In contrast, expression of the antiapoptotic Bcl-2 protein could not be detected in SMMC7721-ICN cells. These findings suggest that Notch1 signaling may participate in the development of HCC cells, affecting multiple pathways that control both cell proliferation and apoptosis.     

2-Methoxyestradiol and paclitaxel have similar effects on the cell cycle and induction of apoptosis in prostate cancer cells

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with promise for cancer chemotherapy, including advanced prostate cancer. We have focused on events related to cell cycle arrest (G1 and G2/M) and induction of apoptosis in human prostate cancer cells. Treatment with 2-ME increased cyclin B1 protein and its associated kinase activity followed by later inhibition of cyclin A-dependent kinase activity and induction of apoptosis. Similar results were obtained with paclitaxel (taxol), a clinically relevant agent used to treat advanced prostate cancer. Cyclin-dependent kinase inhibitors prevented 2-ME and paclitaxel-mediated increase in cyclin B1-dependent kinase activity and blocked induction of apoptosis. Reduction of X-linked inhibitor of apoptosis (XIAP) protein by 2-ME and paclitaxel correlated with increased apoptosis. Lower doses of 2-ME and paclitaxel resulted in G1 (but not G2/M) cell cycle arrest in the p53 wild type LNCaP cell line, but with minimal induction of apoptosis. We suggest that 2-ME and paclitaxel-mediated induction of apoptosis in prostate cancer cells requires activation of cyclin B1-dependent kinase that arrests cells in G2/M and subsequently leads to the induction of apoptotic cell death.     

Efficacy and mechanism of action of the proteasome inhibitor PS-341 in T-cell lymphomas and HTLV-I associated adult T-cell leukemia/lymphoma

HTLV-I associated adult T-cell leukemia (ATL) and HTLV-I-negative peripheral T-cell lymphomas are associated with poor prognosis. Using pharmacological concentrations of the proteasome inhibitor PS-341, we demonstrate inhibition of cell proliferation and induction of apoptosis in fresh ATL cells, HTLV-I transformed and HTLV-I-negative malignant T cells, while normal resting or activated T lymphocytes were resistant. Combination of PS-341 and doxorubicin or etoposide resulted in an additive growth inhibition. In HTLV-I-negative malignant cells, PS-341 treatment significantly downregulated the antiapoptotic protein X-IAP and to a lesser extent c-IAP-1 and bcl-X(L) and resulted in caspase-dependent apoptosis. In HTLV-I transformed cells, the inhibition of the proteasomal degradation of Tax by PS-341 likely explains the relative protection of HTLV-I infected cells against caspase-dependent apoptosis. PS-341 treatment of these cells stabilized IkappaBalpha, IkappaBbeta, IkappaBvarepsilon, p21, p27 and p53 proteins and selectively inhibited Rel-A DNA binding NF-kappaB complexes. In both HTLV-I-positive and -negative cells, PS-341 treatment induced ceramide accumulation that correlated with apoptosis. We conclude that PS-341 affects multiple pathways critical for the survival of HTLV-I-positive and -negative malignant T cells supporting a potential therapeutic role for PS-341 in both ATL and HTLV-I-negative T-cell lymphomas, whether alone or in combination with chemotherapy.     

Up-regulation of the proapoptotic mediators Bax and Bak after adenovirus-mediated p53 gene transfer in lung cancer cells.

Overexpression of wild-type p53 in cancer cells by adenovirus-mediated p53 gene transfer can result in the induction of apoptosis. To identify the potential mediators of this p53-induced apoptosis, we examined apoptotic protein levels in human lung cancer cells after Adp53 gene transfer. We observed up-regulation of Bax and Bak protein levels 18-36 h after transduction with Adp53 in H1299, H358, and H322 lung cancer cells. Contrary to expected observations, no changes in Bcl-2 and Bcl-X(L) protein levels were observed. Morphological cell changes and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining showed evidence of apoptosis in all cell lines 48 h after transduction with Adp53. These results indicate that the induction of apoptosis by adenovirus-mediated p53 transfer may be mediated by the induction of proapoptotic mechanisms rather than suppression of antiapoptotic mechanisms.     

A novel apoptotic mechanism of genetically engineered adenovirus-mediated tumour-specific p53 overexpression through E1A-dependent p21 and MDM2 suppression

Oncolytic viruses engineered to replicate in tumour cells but not in normal cells could be used as tumour-specific vectors carrying the therapeutic genes. We previously developed a telomerase-specific oncolytic adenovirus, OBP-301, that causes cell death in human cancer cells with telomerase activities. Here, we further modified OBP-301 to express the wild-type p53 tumour suppressor gene (OBP-702), and investigated whether OBP-702 induces stronger antitumour activity than OBP-301. The antitumour effect of OBP-702 was compared to that of OBP-301 on OBP-301-sensitive (H358 and H460) and OBP-301-resistant (T.Tn and HSC4) human cancer cells. OBP-702 suppressed the viability of both OBP-301-sensitive and OBP-301-resistant cancer cells more efficiently than OBP-301. OBP-702 caused increased apoptosis compared to OBP-301 or a replication-deficient adenovirus expressing the p53 gene (Ad-p53) in H358 and T.Tn cells. Adenovirus E1A-mediated p21 and MDM2 downregulation was involved in the apoptosis caused by OBP-702. Moreover, OBP-702 significantly suppressed tumour growth in subcutaneous tumour xenograft models compared to monotherapy with OBP-301 or Ad-p53. Our data demonstrated that OBP-702 infection expressed adenovirus E1A and then inhibited p21 and MDM2 expression, which in turn efficiently induced apoptotic cell death. This novel apoptotic mechanism suggests that the p53-expressing OBP-702 is a promising antitumour reagent for human cancer and could improve the clinical outcome.     

The mechanisms of death of an erythroleukemic cell line by p53: involvement of the microtubule and mitochondria

A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. These cells showed typical characteristics of apoptosis, when they were cultured at 32 degrees C. In this process, p53 recovered the wild-type p53 function and the expression of the p21 (waf1/cip1/sdi1), cyclin G1 and gadd45 genes was increased. However, no significant changes were detected in the expression of the mdm2, bcl-2, bax, fas and fasl genes, suggesting the existence of other genes associated with apoptosis. Genes up-regulated by p53 were screened by the mRNA differential display method. One of the up-regulated genes was identified as the elongation factor 1 alpha (EF-1 alpha) gene. EF-1 alpha is also a microtubule-severing protein. Upon the temperature-shift, the cells developed the morphology and the localization of alpha-tubulin similar to those of the cells treated with vincristine, a drug that affects microtubules. The microtubule-severing associated with up-regulation of EF-1 alpha by p53 may be a cause of the cell death. On the other hand, the function of cyclin G1 is not so clear despite the fact that 1-2-3 cells showed a significant increase of the cyclin G1 gene during the early stage of apoptosis. The yeast two-hybrid system was used to identify cyclin G1-associated proteins. One is a cytochrome c (Cyt c) oxidase subunit II (COXII). Cyclin G1 and COXII were co-immunoprecipitated from an extract of human osteosarcoma cell line that expressed high levels of cyclin G1. COX activity was also increased by temperature-shift in this cell line. The pattern of changes in COX activity was closely reflected by the expression of the cyclin G1 gene. Cyclin G1 and COXII associate physically with each other in vivo and that activation of COXII by binding to cyclin G1 upregulated by p53 may be associated with apoptosis. These two new pathways, p53-EF-1 alpha-microtubule-severing (-distortion of cytoskeleton) and p53-cyclin G1-COXII (-CytC, ATP-caspase-3 activation), may cooperate to induce apoptosis in this cell line.     

p19ARF prevents G1 cyclin-dependent kinase activation by interacting with MDM2 and activating p53 in mouse fibroblasts.

p19ARF encoded by the INK4a tumor suppressor gene locus functions upstream of p53 to induce cell cycle arrest. p19ARF can interact with MDM2 and p53 in cells ectopically overexpressing these three components, but the biochemical cascades from p19ARF to cell cycle arrest has not been fully elucidated. In this study, we generated stably transfected NIH3T3 cells that express exogenous p19ARF under the control of a heavy metal-inducible metalothionine promoter. Cells arrested in G1 by ectopically expressed p19ARF contained considerably reduced G1 cyclin dependent kinase (cdk2 and cdk4) activities. The expression of cyclin A (a regulatory subunit of cdk2) markedly decreased, while cyclin D1, the major cdk4 partner in fibroblasts, expressed at a slightly higher level and formed complexes with cdk2 and cdk6 in addition to cdk4. Induction of p19ARF activated p53 by increasing its stability, and allowed the expression of p21Cip1, which bound to all of the cyclin D1-cdk complexes (cyclin D1-cdk2, -cdk4, and -cdk6) thereby inhibiting their kinase activities. p19ARF formed complexes with several cellular proteins including mouse MDM2. The majority of MDM2 was found in the complex with p19ARF, while no p53 was detected in association with p19ARF. Thus, we propose that p19ARF neutralizes MDM2 by sequestration from p53, which results in activation of p53, inhibition of G1 cyclin-cdk activities, and G1 arrest.     

Human tumor suppressor ARF impedes S-phase progression independent of p53

Using alternative reading frames, the human ARF-INK4a locus encodes two unrelated proteins that both function in tumor suppression. p16(INK4a) maintains the retinoblastoma protein in its growth-suppressive state through inhibition of cyclin D-dependent kinase activity, whereas ARF binds with MDM2 and stabilizes p53. The majority of the activity of ARF to date is ascribed to its ability to activate p53, resulting in a G(1) cell cycle arrest or apoptosis. We show here that ARF colocalizes with DNA replication protein A (RPA32) and that overexpression of ARF reduces the rate of DNA synthesis resulting in accumulation of an S-phase cell population. Impediment of DNA synthesis by ARF can occur and becomes more evident in the absence of p53. Hence, the biological consequence of ARF induction varies dependent on cellular p53 status, inducing predominantly a G(1) arrest or apoptosis in p53-positive cells or causing S-phase retardation when p53 function is comprised.