Multiple domains of the mouse p19ARF tumor suppressor are involved in p53-independent apoptosis

The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.     

p19ARF-induced p53-independent apoptosis largely occurs through BAX

Combined disruption of the ARF gene and the p53 gene causes mouse predisposition to tumors of a wider variety and at a higher frequency than disruption of the p53 gene, indicating that the ARF gene has p53-independent anti-tumor function in addition to p53-dependent function. Coincidentally with this notion, ectopic expression of the p19(ARF) induces apoptosis for wild-type mouse embryo fibroblasts which have been immortalized by introduction of the SV40 virus genome (SV40-MEFs). The protein expression levels of p53, p21(Cip1), and Bax were not upregulated by ectopic expression of p19(ARF) in SV40-MEFs, indicating that expression of p19(ARF) induced apoptosis through p53-independent pathways in this system. Ectopic expression of p19(ARF) induced prominent apoptosis even in SV40-Bak-/-MEFs. In contrast, expression of p19(ARF) induced only a very low grade of apoptosis in Bax-/- or Bax-/-/Bak-/-SV40-MEFs. Remarkable attenuation of p19(ARF)-induced apoptosis by disruption of the Bax gene thus leads to the conclusion that Bax plays a major role in p53-independent apoptosis induced by p19(ARF).     

p53-independent apoptosis in UV-irradiated mouse skin: possible inhibition by 50 Hz magnetic fields

Our recent results suggest that 50 Hz magnetic fields (MF) enhance ultraviolet (UV)-induced tumorigenesis in mouse skin. The aim of the present experiment was to study suppression of apoptosis as a possible mechanism for MF effects on skin tumorigenesis. Another aim was to test the importance of a UV and MF exposure schedule, particularly the role of MF exposure prior to UV irradiation. Female mice were exposed to a UV dose of 2 human MED and to 100 microT MF of 50 Hz, using the following exposure schedules: group 1 sham MF 24 h, UV 1 h, sham MF 24 h; group 2 sham MF 24 h, UV 1 h, MF 24 h; group 3 MF 24 h, UV 1 h, MF 24 h. Lamps emitting simulated solar radiation (SSR) were used for UV irradiation. Skin samples were analysed for apoptosis, expression of the p53 gene, activity of the enzyme ornithine decarboxylase (ODC) and polyamine concentrations. A significantly (p = 0.017) lower number of apoptotic cells was measured in group 2 compared to group 1. A similar but not statistically significant (p = 0.064) decrease was also detected in group 3. No p53 expression was detected in any sample. The levels of ODC and putrescine did not differ significantly between the UV-only and UV and MF-exposed groups. Spermidine and spermine levels were significantly (p = 0.014 and 0.014, respectively) lower in group 3 than in group 1, but no decrease was observed in group 2. Our findings suggest that SSR induces p53-independent apoptosis in mouse skin and that the apoptotic response may be inhibited by exposure to MF. The exposure schedule did not alter the MF effect. The results do not support a causal role for polyamines in MF effects on apoptosis.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway

Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers.     

Pituitary tumor transforming gene causes aneuploidy and p53-dependent and p53-independent apoptosis

The pituitary tumor transforming gene, PTTG, is abundantly expressed in several neoplasms. We recently showed that PTTG overexpression is associated with apoptosis and therefore have now studied the role of p53 in this process. In MCF-7 breast cancer cells that express wild type p53, PTTG overexpression caused apoptosis. p53 was translocated to the nuclei in cells expressing PTTG. Overexpression of p53, along with PTTG, augmented apoptosis, whereas expression of the human papillomavirus E6 protein inhibited PTTG-induced apoptosis. In MG-63 osteosarcoma cells that are deficient in p53, PTTG caused cell cycle arrest and subsequent apoptosis that was inhibited by caspase inhibitors. A proteasome inhibitor augmented PTTG expression in stable PTTG transfectants, suggesting that down-regulated PTTG expression is required for cell survival. Finally, MG-63 cells expressing PTTG showed signs of aneuploidy including the presence of micronuclei and multiple nuclei. These results indicate that PTTG overexpression causes p53-dependent and p53-independent apoptosis. In the absence of p53, PTTG causes aneuploidy. These results may provide a mechanism for PTTG-induced tumorigenesis whereby PTTG mediates aneuploidy and subsequent cell transformation.     

Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis

Doxorubicin (DOX) is a common anticancer drug. The mechanisms of DOX induced apoptosis and the involvement of reactive oxygen species (ROS) in apoptotic signaling were investigated in p53-null human osteosarcoma Saos-2 cells. Accumulation of pre-G1 phase cells and induction of DNA laddering, which are the hallmarks of apoptosis, were detected in cells at 48 h upon DOX treatment. Furthermore, DOX increased the intracellular hydrogen peroxide and superoxide levels, followed by mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, prior to DNA laddering in Saos-2 cells. In addition, DOX treatment also upregulated Bax and downregulated Bcl-2 levels in the cells. The role of ROS in DOX induced cell death was confirmed by the suppression effect of catalase on DOX induced ROS formation, mitochondrial cytochrome c release, procaspase-3 cleavage, and apoptosis in Saos-2 cells. The catalase treatment however only suppressed DOX induced Bax upregulation but had no effect on Bcl-2 downregulation. Results from the present study suggested that ROS might act as the signal molecules for DOX induced cell death and the process is still functional even in the absence of p53.     

The p53-inducible E3 ubiquitin ligase p53RFP induces p53-dependent apoptosis

Recently, it has been shown that really interesting new gene (RING)-in between ring finger (IBR)-RING domain-containing proteins, such as Parkin and Parc, are E3 ubiquitin ligases and are involved in regulation of apoptosis. In this report, we show that p53-inducible RING-finger protein (p53RFP), a p53-inducible E3 ubiquitin ligase, induces p53-dependent but caspase-independent apoptosis. p53RFP contains an N-terminal RING-IBR-RING domain and an uncharacterized, evolutionally highly conserved C-terminal domain. p53RFP interacts with E2 ubiquitin-conjugating enzymes UbcH7 and UbcH8 but not with UbcH5, and this interaction is mediated through the RING-IBR-RING domain of p53RFP. Interestingly, the conserved C-terminal domain of p53RFP is required and sufficient for p53RFP-mediated apoptosis, suggesting p53RFP-mediated apoptosis does not require its E3 ubiquitin ligase activity. Together with a recent report showing that p53RFP is involved in ubiquitination and degradation of p21, a p53 downstream protein promoting growth arrest and antagonizing apoptosis, our findings suggest that p53RFP is involved in switching a cell from p53-mediated growth arrest to apoptosis.     

Camptothecin induces p53-dependent and -independent apoptogenic signaling in melanoma cells

Various DNA-targeting agents may initiate p53-dependent as well as p53-independent response and subsequent apoptosis via alternative cellular systems which include for instance p73, caspase-2 or Bcl-2 family proteins. The scope of involvement of individual molecules in this process and the mechanisms governing their potential interplay are still not entirely understood, in particular in highly aggressive cancers such as in malignant melanoma. In this work we investigated the role and involvement of both p53-dependent and -independent mechanisms in selected melanoma cell lines with differing status of p53 using a model DNA topoisomerase I inhibitor camptothecin (CPT). Here we report that CPT induced in Bowes melanoma cells apoptosis which is essentially p53 and mitochondria-dependent but with some involvement of caspase-2 and p73. Conversely, in mutant p53 melanoma cells overall levels of CPT-induced apoptosis are significantly lower, with p73 and caspase-2 signaling playing important roles. In addition, in these cells the expression of micro RNAs family 34 (miR-34) were low compared to wild-type p53 cells. The ectopic expression of wild type p53 than restored apoptotic response of cells to CPT despite the fact that the expression of miR-34 and miR-155 were not influenced. These results suggest that CPT induces multivariate cellular stress responses including activation of DNA-damage response-p53 pathway as well as p53-independent signaling and their mutual crosstalk play the decisive role in the efficient triggering of apoptosis in melanoma cells.     

Triad 1 induces apoptosis by p53 activation

Triad 1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1) is an E3 ligase that induces apoptosis and clonogenic inhibition in myeloid cells through Gfi-1 stabilization. Here we demonstrate that Triad 1 induces apoptosis in several cancer cell lines including MCF7, A549, U2OS, and HCT 116 p53^+/+ cells via its RING ligase activity. Interestingly, in these cancer cells, Triad 1-induced apoptosis is not mediated by Gfi-1 stabilization but is instead p53-dependent. Moreover, Triad 1 promotes transactivation of p53. These results suggest that Triad 1 can induce apoptosis through its ligase activity via p53 activation.     

ZBP-89-induced apoptosis is p53-independent and requires JNK

ZBP-89 induces apoptosis in human gastrointestinal cancer cells through a p53-independent mechanism. To understand the apoptotic pathway regulated by ZBP-89, we identified downstream signal transduction targets. Ectopic expression of ZBP-89 induced apoptosis through the mitochondrial pathway and was accompanied by activation of all three MAP kinase subfamilies: JNK1/2, ERK1/2 and p38 MAP kinase. ZBP-89-induced apoptosis was markedly enhanced by ERK inhibition with U0126. In contrast, inhibiting JNK with a JNK1-specific peptide inhibitor or dominant-negative JNK2 expression abrogated ZBP-89-mediated apoptosis. The p38 inhibitor SB202190 had no effect on ZBP-89-induced cell death. Protein dephosphorylation assays revealed that ZBP-89 activates JNK via repression of JNK dephosphorylation. Oligonucleotide microarray analyses revealed that ectopic expression of ZBP-89 downregulated expression of the dual-specificity phosphatase MKP6. Overexpression of MKP6 blocked ZBP-89-induced JNK phosphorylation and PARP cleavage. In addition, ectopic expression of ZBP-89 repressed Bcl-xL and Mcl-1 expression, but had no effect on Bcl-2. Silencing ZBP-89 with small interfering RNA enhanced both Bcl-xL and Mcl-1 expression. Taken together, ZBP-89-mediated apoptosis occurs via a p53-independent mechanism that requires JNK activation.     

A dominant negative form of p63 inhibits apoptosis in a p53-independent manner

Stem cells are a source of differentiated cells in multiple tissues. If genetic alterations occur in stem cells, the problem persists and malignant cancers may arise. DeltaNp63alpha-a homologue of the tumor suppressor p53-is exclusively expressed in proliferating undifferentiated epithelial cells and cancer cells of epidermal origin. Here, we show that DeltaNp63alpha antagonizes DNA damage-induced apoptosis in a p53-independent manner. We found that upon cellular injury, DeltaNp63alpha must be downregulated before apoptotic program can be activated. The 5637 cell line has abundant levels of DeltaNp63alpha and mutant p53, and it is resistant to DNA damage-induced apoptosis. The knockdown of DeltaNp63alpha by RNA interference sensitized these cells to apoptosis upon genotoxic insult. This suggests that DeltaNp63alpha plays an anti-apoptotic role regardless of the p53 status. Considering the frequent mutations of p53 in tumor cells, our results provide important implications for the treatment of cancers in which p63 is amplified.     

Influence of abdominal temperature and luminal contents on the cauda epididymidis of the rat

In an attempt to determine if changes previously described in the epididymides of cryptorchid testes were related to the elevated environmental temperature or to the absence of normal luminal constituents, rats were divided into four test groups. Group I animals were made unilaterally cryptorchid. Animals in Group II had only the cauda epididymidis of one side maintained within the abdominal cavity (cryptepididymal) while the caput epididymides and testes remained in the scrotum. The testes of animals in Group III remained in the scrotum but had their efferent tubules ligated on one side. Testes of unoperated rats and contralateral testes of the test animals served as controls. The histochemical demonstration of sorbitol dehydrogenase (SDH) was used to determine differences in functional activity and light and electron microscopy were used to determine structural changes. SDH activity could not be demonstrated in the cauda epididymidis of cryptorchid and efferent tubule-ligated animals; animals in which the luminal contents were obviously changed. These same groups of animals showed abnormal folding of the basal surface of the epididymal epithelium at the ultrastructural level. Activity of SDH could be demonstrated in control epididymides and in those that contained normal luminal contents but were maintained at the temperature of the abdominal cavity. The basal surface of the epididymal epithelium was not unusual in these animals. The results indicate that the epididymis is influenced to a greater extent by changes in luminal contents than by temperature elevation.     

Adenoviral Bid overexpression induces caspase-dependent cleavage of truncated Bid and p53-independent apoptosis in human non-small cell lung cancers

Proapoptotic gene transfer to promote death or to augment killing by DNA-damaging agents represents a promising strategy for cancer therapy. We have constructed an adenoviral Tet-Off trade mark vector with tightly controlled expression of Bid (Ad-Bid) (Clontech, Palo Alto, CA). Using the non-small cell lung cancer cell lines H460, H358, and A549, low dose Ad-Bid was shown to induce high levels of full-length Bid as well as caspase-3 and -9 activity. Although only a small fraction of Bid was processed to truncated Bid (a step inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), Ad-Bid gene transfer resulted in mitochondrial changes consistent with apoptosis (mitochondrial depolarization, cytochrome c release), DNA fragmentation, and a dramatic loss of cell viability. The proapoptotic effects of Ad-Bid were independent of p53 status and were augmented markedly by caspase-8 activators such as the DNA-damaging agent cisplatin. When Ad-Bid and cisplatin were used together, chemosensitivity was restored in p53-null H358 cells, increasing death from 35% following treatment with cisplatin and Ad-LacZ to >90% death with Ad-Bid and cisplatin (Ad-Bid alone induced 50% cell death under these conditions). Ad-Bid can induce apoptosis in malignant cells and enhance chemosensitivity in the absence of p53, suggesting this approach as a potential cancer therapy.     

Oncogenic H-ras induces cyclin B1 expression in a p53-independent manner

Alcohol abuse is a major risk factor for cancer of the upper alimentary tract, the upper respiratory tract, and liver. Chromosome damage is used as early effect biomarker in the surveillance of human exposure to genotoxic carcinogens. In the present study, two genetic markers, namely chromosome aberrations (CAs) and micronuclei (MN), were used to evaluate genetic damage in peripheral lymphocytes from 20 alcoholics, 20 abstinent alcoholics, and 20 controls. Composition of the three groups was fairly similar as regards sex, age and smoking habits. A highly significant increase was observed in the frequencies of CA and MN in lymphocytes of alcoholics as compared both with controls and abstinent alcoholics. However, no correlation was found between the length of alcohol abuse and the frequencies of either biomarkers in alcoholics. CA and MN frequencies in abstinent alcoholics were similar than those in controls.Our data indicate that CA and MN can be two useful biomarkers to assess genetic damage associated with alcohol abuse. They could be included in programs for cancer prevention in alcoholics. Abstinence appears to normalize the frequency of both MN and CA. This could offer therapists another tool to help alcoholics change their lifestyle.     

p53-independent pRB degradation contributes to a drug-induced apoptosis in AGS cells

The retinoblastoma （RB） tumor suppressor protein, pRB, plays an important role in the regulation of mammalian cell cycle. Furthermore, several lines of evidence suggest that pRB also involves in the regulation of apoptosis. In the present study, the degradation of pRB was observed in apoptotic gastric tumor cells treated with a new potent anti-tumor component, tripchlorolide （TC）. The inhibition of pRB degradation by a general cysteine protease inhibitor IDAM resulted in the reduction of the apoptotic cells. Furthermore, the survival of the gastric tumor cells under the TC treatment was enhanced by an over-expression of exogenous pRB. These results suggest that the pRB degradation of the gastric tumor cells under the TC treatment involves in the apoptotic progression. In addition, the same extent of TCinduced pRB-degradation was detected in the gastric tumor cells containing a p53 dominant-negative construct, indicat- ing that this kind of pRB degradation is p53-independent.