Effects of PFT-[alpha] on cell survival and transactivation of known p53 responsive genes using rat liver cells and HepG2 cell line.

ABSTRACT

Pifithrin-[alpha] (PFT-[alpha]) is a reversible inhibitor of p53-mediated apoptosis, p53-dependent gene transcription, as well as down stream responsive gene function. In response to genotoxic agents, normal cells are instructed by p53 either to perform DNA repair or to undergo apoptosis. Studies showed that chemo and/or radiotherapy damage both normal and cancerous cells indiscriminately. The objective therefore, was (1) to evaluate PFT-[alpha] for differential cellular protection in response to arsenic trioxide and cadmium chloride exposure of normal and neoplastic cells, and (2) to evaluate the transcriptional activation of p53 and p53-responsive genes in rat liver cells and HepG2 carcinoma cell line. Cells were cultured to 90% confluency and subsequently exposed to the cytotoxic agents in presence or absence of PFT-[alpha] for a 48-hour period. Cell survival was detected by fluorescein diacetate (FDA) and fluorospectroscopy. Percent survival indices (L[C.sub.50]) were calculated using regression analysis. Mean L[C.sub.50] and (SD) for HepG2 cells following exposure to arsenic were 13.7 ([+ or -]1.0) [micro]g/ml with PFT-[alpha] and 13.4 ([+ or -] 0.5) [micro]g/ml without PFT-[alpha] (p>0.05). For rat liver cells it was 670 ([+ or -] 8.15) [micro]g/ml with and 573.15 ([+ or -]1.0) [micro]g/ml without PFT-[alpha] (p<0.05). On exposure to cadmium Chloride, L[C.sub.50]'S were 6.95 ([+ or -]2.5) [micro]g/ml for HepG2 cell line in presence of PFT-[alpha] and 7.35 ([+ or -]1.9) [micro]g/ml in its absence (p>0.5). With rat liver cells exposed to cadmium chloride the L[C.sub.50] was found to be 57.72 ([+ or -]0.8) [micro]g/ml and 58.1 ([+ or -]5.5) [micro]g/ml, (p>0.5), in presence of PFT-[alpha] and in its absence respectively. The results revealed significant differences from controls only upon exposure of rat liver cells to arsenic trioxide in presence of PFT-[alpha]. Since p53 is not responsive to cadmium chloride, results on the transcriptional activation of p53 and p53 responsive genes; PCNA, mdm2, cyclin G, p21, and bc12 were done only in response to arsenic trioxide and were demonstrated using Western blot analysis. PFT-[alpha] inhibited the transactivation of p53 in rat liver cells and resulted in repression of bcl2, PCNA and mdm2, cyclin G and p21 genes by arsenic trioxide. HepG2 cells exposed to arsenic trioxide and PFT-[alpha] showed expression of only p53 and PCNA genes. We conclude that PFT-[alpha] exhibits cytoprotective effect, modifies the detrimental influences of known genotoxic agents in normal cells and has the potential for being used as a beneficial adjuvant to cancer therapy.

INTRODUCTION

The need for chemo and/or radiotherapy in addition to medical and surgical treatment for cancer has been rising side by side with the human life expectancy. This need was concurrent with increases in the number of cancer afflictions where one in every four victims is suffering terminal consequences [1, 2]. Most chemotherapeutic agents have negative toxic side effects on healthy normal cellular survival manifested by nausea, vomiting, diarrhea, chronic anemia, hair and weight losses that limit their efficacy and application. Consequently, an ongoing search has been executed to find agents that have protective effects to normal cells against the adverse side effects of cancer therapies.

Exposure to chemical carcinogens as well as the introduction of foreign viral DNA into cells manifest as fast accumulations of simple local changes in DNA sequence, chromosome breaks, and/or translocations, and such events mostly precede carcinogenesis and cancer progression. The DNA damaging agents, such as gamma-irradiation, UV light and drugs like etoposide, methylmethane sulfate etc. cause spectacular amplification of the p53 gene in cells of susceptible tissues. DNA damage also activates a suite of protein kinases such as ATM (Ataxia Telangiectasia mutant) and DNA-PK. ATM targets p53 protein, a transcription factor that can bind to specific DNA sequences and transactivate certain p53-responsive genes while DNA-PK targets mdm2. Mdm2 is a p53 downstream-responsive gene that binds to p53 and maintains it at low levels through degradation during non-stressed normal growth of cells, thus protecting cells from growth arrest and apoptosis. As a transcription factor, mdm2 blocks the ability of p53 to perform nuclear transcription of other genes [3, 4, 5, 6, 7, and 8].

DNA damage results in p53 activation, mdm2 down-regulation and consequently three cellular responses are bound to follow namely, growth arrest at [G.sub.1], DNA repair, or apoptosis. Moderate elevation of p53 induces production of a 37 kDa; PCNA, a well-conserved protein in both mammalian and plant cells, to help in DNA repair. Transcription of p21 is activated by p53 in order to arrest progression through cell cycle allowing cellular machinery to repair its damaged DNA. The p53 gene regulates the repression of the antiapoptotic bcl2 family, which guides those cells who are beyond repair into apoptosis There are also events that involve p53 dependent repression of anti-apoptotic genes of the bcl2 family that are involved in pro-survival and interfere with apoptosis. [9, 10, 11, and 12]

The first report on Pifithrin-[alpha] (PFT-[alpha]; 1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H) benzothiazoyl ethanone.HBr, with a [C16H18N2OS.HBr] chemical structure) was published in 1999. It is a synthetic water-soluble stable molecule with a molecular weight of 367. The chemical was established as a novel compound that protected mice against side effects of radiation. Following 24 hours exposure, PFT-[alpha] attenuated [beta]-gal induction in doxorubicin (Dox) and UV light exposed cells in a dose dependent manner without affecting cell growth or cell survival. PFT-[alpha] inhibited apoptotic death of C8 cells, a conventional model for p53 dependent apoptosis induced by DOX, etoposide, taxol, UV light and [gamma]-irradiation [13].

Recent studies on the use of PFT-[alpha] included protection of mice from the side effects of radiation and chemotherapy of cancer [3, 14, and 15] as well as the role of p21 on the induction of p53-dependent and independent apoptosis in the support for chemoprevention of colon cancer [16].

A p53-independent Fas-mediated apoptosis in MCF-7 breast cancer cells in response to sodium butyrate was determined using the PFT-[alpha] [17] as well as the increased induction of genetic instability in lymphblastoma cells by etoposide (VP16) [18]. Objectives such as p53-dependent PFT-[alpha] regulation of hepatocyte proliferation in rats [19], the acceleration of cutaneous wound healing by the inhibitor through the inhibition of p53 function [20] and the protective role of the PFT-[alpha] in the inhibition of ischemic-reperfusion were addressed [21].

The roles of PFT-[alpha] in the suppression of double strand break repairs in mammalian chromosomes [22], in the regulation of cell survival, apoptosis and growth arrest by p53 and polymerase [23], and in the suppression of heat shock and gluocorticoid signaling pathways have also been researched [24].

The effects of PFT-[alpha] in protecting liver tissue against endotoxin-induced apoptotic and necrotic cell death [25], as well as the role of the inhibitor on the promotion of p53-mediated apoptosis in JB6 cells were determined [26].

UV-induced apoptotic response in CS-B cells was unaffected by PFT-[alpha]-induced inhibition of p53, p21 and Bax [27]. The roles of this inhibitor in protecting mice from DOX-induced apoptosis in acute cadiotoxicity [28] and the inhibition of p53 signaling after interaction with heat shock protein 90 (HSP90) and its nuclear translocation were established[29] and the improvement of rat liver graft quality by PFT-[alpha]-mediated inhibition of hepatocyte necrapoptosis was also determined [30].

The susceptibility to PFT-[alpha] helped to create an interval in the cell cycle for the purpose of DNA repair and thus increasing the percentage of survival in normal cells. The P53-dependent transactivation of target genes including cyclin G, P21/wafl/Cipl/mdm2 underwent reversible inhibition by PFT-[alpha] as judged by Northern Blot analysis [13]. The influence of PFT-[alpha] on DNA synthesis at the tissue level following whole body [gamma]-irradiation, as assessed by [.sup.14.C] thymidine incorporation assay, in mice showed proportional changes in thymidine incorporation. The extent of apoptosis in gut epithelium of [gamma]-irradiated mice established that PFT-[alpha] attenuated p53 dependent blockage of DNA replication in rapidly proliferating tissues. With these encouraging results, the cancer research community began to harbor great expectations in the efficacy of PFT-[alpha] and other compounds with similar properties in the reduction/or alleviation of side effects of radiation or chemotherapy in human cancers. The temporary suppression of p53 gene function might help reduce the side effects of anti-cancer regimens or even results in their complete elimination.

Inorganic arsenic is a recognized human carcinogen [31-38]. Cadmium was classified as a human carcinogen by IARC. We chose arsenic trioxide and cadmium chloride as chemical inducers with the understanding that almost all chemicals of environmental interest that induce p53 could cause DNA damage [39]. Arsenite was found to interfere with DNA repair and that the in vitro arsenic-induced apoptosis is dependent on its dose, type cell and environmental factors. In addition, arsenic as a carcinogen acts by inducing DNA hypomethylation to compliment devious gene expressions, and thus was regarded as a cocarcinogen or promoter of carcinogenesis [40].

The study's goal was to evaluate the mammalian cell response to toxic agents & differential protection by PFT-[alpha] and thus included the following: Objective (1) to establish the degree of cytotoxicity induced by arsenic and cadmium on HepG2, rat liver cells, and to assess the protective effect of PFT-[alpha]. Objective (2) to establish the role of arsenic trioxide in transactivation of p53-responsive genes in relation to PFT-alpha protection and objective (3) to provide basis for elucidating the potential mechanisms involved in arsenic/cadmium-induced hepatotoxicity.

It was intended to examine the hypothesis that there is a degree of protection to mammalian cells offered by the p53 inhibitor (PFT-[alpha]) from toxicity induced by two chemical carcinogens (arsenic trioxide and cadmium chloride) and being differential between the two liver cell types. This work will attempt to examine the inactivation trends of the p53 gene by PFT-[alpha], its dose response kinetics, the manner in which PFT-[alpha] affects transactivation of known p53 responsive genes such as p21, PCNA, Cyclin G, mdm2 and bcl2. It is expected that the findings will provide additional information on potential mechanisms involved in toxicity induced by DNA damaging agents as well as providing basis for arsenic and cadmium induced hepatotoxicity. The study was also aimed at suggesting development of clinical applications and novel therapeutic strategies towards alleviating the potentially detrimental side affects of anti-cancer agents.

MATERIALS AND METHODS

HepG2 tumor cells are a continuous cell line obtained from neoplastic tissue. Morphologically they are epitheloid and originated from adult human hepatoma. The cells are aneuploid with some microsomal metabolizing enzymes [41]. This cell line was purchased from the American Type Culture Collection (ATCC). The normal rat liver cells were prepared in our laboratory from a male Wistar rat [42]. The rat was sacrificed after it had been anaesthetized and liver was removed under strict sterile conditions. Excised hepatic tissue was shredded under sterile conditions, suspended in Accumax, a trypsin substitute enzyme (Innovation Cell Technologies, San Diego, California), solution (lml/lmg of tissue), and placed on the heater rocker at 37[degrees]C for 15 mins. The cell suspension was centrifuged at 3000 rpm for 5 minutes followed by complete resuspension of cell pellet in small amount of fresh DMEM (Dulbecco's Modified Eagle's Minimal Essential Medium, life Technologies in Grand Island, New York); supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin. Tissue culture flasks with complete medium were seeded with inoculum and incubated at 37[degrees]C in a C[O.sub.2] in incubator.

Vials containing the HepG2 cells (cryosafe preserved) were thawed by gentle agitation for 2 minutes in a water bath at 37[degrees]C. Preparation of cell cultures followed the above-mentioned inoculation and incubation protocols. On reaching about 90% confluency, both cells were washed with phosphate buffered saline (PBS), trypsinized with 10 mL of 0.25% (w/v) trypsin-0.03% (w/v) EDTA, diluted with fresh medium, counted, and seeded (2.5-5 x [10.sup.5] cells/ml) in two sets of 96-well microtiter tissue culture plates for cytotoxicity determination. To the designated plate rows, 180 [micro]l of fresh medium containing 10mM of Pifithrin-[alpha] [2-(2-Imino-4, 5,6,7 tetrahydrobenzothiazol-3-yl)-l-p-tolylethanone-HBr (CN Biosciences, Inc., San Diego, California)].was added to each well. Twenty (20[micro]l) of serial dilutions of cadmium chloride (Cd[Cl.sub.2], Fisher Scientific, Fair Lawn, New Jersey). Chemical concentrations of 0, 1.9, 3.9, 7.8, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 [micro]g/ml) and arsenic trioxide (0, 0.19, 0.39, 0.78, 1.562, 3.125, 6.25, 12.5, 25, 50, 100 and 1000 [micro]g/ml were prepared. Aliquots were added columnwise separately to each of the 96-well plates with cells and incubated for 48 hours. The medium in each well was removed, wells were washed with 200 [micro]l of cold PBS, and then 100[micro]l FDA (Fluorescein Diacetate; Molecular Probes Inc, Eugene, Oregon) working solution (10 [micro]g/ml in PBS) was added columnwise to each well and incubated for 1 hr. Surviving cells hydrolyzed the added non-fluorescent FDA to fluorescein which was measured with Fluoroskan Ascent FL (Thermo Labsystems Inc) at excitation/emission wavelengths of 346/432 nm. The values obtained per concentration were converted to percentage cell viability. Data comprised four replicates each of three different repeated experiments. Regression analysis was performed on percent cell viability data and from the resulting equations, the lethal concentration needed to kill 50 % of the cells (L[C.sub.50]) were computed.

Western blots were carried out to demonstrate p53 transactivation of p21, PCNA, Cyclin G, mdm2 and bcl2 genes for both rat liver cells and Hep G2 cell line. The protein extracts from normal rat liver cells and HepG2 cell line exposed to arsenic trioxide only were used for western blotting because results of survival assays indicated that cadmium chloride induced toxicity was possibly not p53 mediated. The preparation of cell cultures followed the above-mentioned inoculation and incubation protocols. On reaching about 90% confluency, both cells were washed with phosphate buffered saline (PBS), trypsinized with 10 mL of 0.25% (w/v) trypsin-0.03% (w/v) EDTA, diluted with fresh medium, counted and seeded (2.5-5 x [10.sup.5] cells/ml) in marked tissue culture dishes. Categories were, control cells, cells exposed to PFT-[alpha] only, cells exposed to arsenic trioxide only, cells exposed to PFT-[alpha] and arsenic trioxide. After 48 hours of incubation at 37[degrees]C, cell lysates were collected from each plate separately and total protein determination was done using the Bradford assay methodology. Express gel [GENE Mate] was used for the separation of proteins by electrophoresis (using Precast Polyacrylamide Mini gels; ISC Bio Express, Kaysville, Utah), running buffer, SDS-PAGESDS-PAGE and Western blotting cell unit (from BIORAD). Bands were transferred to membranes by the use of Scotch brite Fiber pads, nitrocellulose membrane, filter papers cut to size, and transfer buffer. Blocking of nonspecific reaction was done with 5% blocking solution (5% non-fat dry milk). The cells were washed, lysed, and cell lysates were incubated with primary antibody followed by washing and incubation with the secondary antibody [43]. This process was followed by incubation with detection reagents for developing with Anti-IgG (AP Conjugate). The AP buffer was used for Immunodetection by the alkaline phosphatase method [44]. NBT [Nitro Blue Tetrazolium] solution BCIP [5-Bromo-4 Chloro-3-indolyl phosphate], 33 /10 ml of AP buffer and scanned with Nucleotech Nucleovision apparatus. Beta actin standard was used for equal loading of samples. Statistics were performed using descriptive and linear regression analyses.

RESULTS AND DISCUSSION

The differential patterns of survival of rat liver cells and HepG2 cells following 48 hour-exposure to the p53 inhibitor was shown in Fig 1. As we can see, the survival for both cell types was followed by a gradual decrease in viability due to arsenic trioxide and PFT-[alpha] exposure in a dose-dependent fashion (Fig 1). The same trend was observed at considerably lower exposures of cadmium chloride (data not shown).

The toxicity indices obtained by regression analyses (the linear part of the survival curve), depicting the manner in which rat liver cells were affected following 48 hr exposure to arsenic trioxide alone or PFT-[alpha] were shown in table 1. The L[C.sub.50] for the HepG2 cell line exposed to arsenic trioxide were 13.7 [+ or -] 1.0 [micro]g/ml and 13.4 [+ or -] 0.6 [micro]g/ml with and without PFT-[alpha] respectively. The L[C.sub.50]'S obtained for the rat liver cells showed a similar trend to those obtained for the Hep[G.sub.2] cell line. A regression value ([R.sup.2]) of 1.0 was obtained for PFT[alpha] and was 0.91 in the absence of PFT-[alpha]. Survival indices for rat liver cells (L[C.sub.50]) were 670 [+ or -] 8.15 [micro]g/ml and 573 [+ or -] 26.2 [micro]g/ml for arsenic trioxide with and without PFT-[alpha] respectively. A similar L[C.sub.50] for arsenic trioxide on HepG2 cells has been previously reported [37].

On exposure to cadmium chloride, the observed trend for normal rat liver cells was that the [R.sup.2] was 0.80 with PFT-[alpha] and 0.79 without PFT-[alpha] respectively. The corresponding L[C.sub.50]'S were 57.72 [+ or -] 0.8 [micro]g/ml and 58.1 [+ or -] 5.5 [micro]g/ml respectively (Table 1). The cytotoxic effect of cadmium chloride on Hep[G.sub.2] cell line following 48 hrs exposures with and without PFT-[alpha] was also shown in table 1 and fig 2. These findings also depict strong dose-response relationship; [R.sup.2] being 0.93 with PFT-[alpha] and 0.93 without PFT[alpha]. The respective L[C.sub.50s] values were 6.95 [+ or -]2.5 [micro]g/ml and 7.35 [+ or -] 1.9 [micro]g/ml.

In reference to table 1 regarding the L[C.sub.50] of rat liver cells exposed to arsenic trioxide, this indicator showed that the L[C.sub.50] has improved almost a 100 units with addition of PFT-[alpha] (p<0.05). The rat liver cells exposed to cadmium chloride showed no difference (L[C.sub.50] remained the same for both PFT-[alpha] exposed and none exposed cells). From this comparative representation, we can assume that growth arrest and programmed cell death in response to arsenic trioxide in rat liver cells are p53 dependent. This effect was absent in exposed HepG2 cells due to the presence of mutated p53 in these cells. The response to cadmium chloride in both rat liver cells and HepG2 cells were apparently p53-independent because PFT-[alpha] did not change the L[C.sub.50] either positively or negatively in both cell types. It is worth mentioning, based on the toxicity indices obtained in this study, that arsenic trioxide was far less toxic in comparison to cadmium chloride. It was also shown that the normal cells were at an average of 46 and 8 times more resistant than the neoplastic cell line respectively for arsenic and cadmium, pointing to the deranged metabolic status in cancer cells (Fig 2).

The cytotoxic effects observed by exposing rat liver cell to arsenic trioxide depicted different observations; PFT-[alpha] did provide protection to rat liver cells upon exposure to arsenic trioxide: L[C.sub.50s] were 670 [+ or -] 8.15 [micro]g/ml and 573 [+ or -] 26.2 [micro]g/ml with and without PFT-[alpha] respectively (Fig 2). Our findings concerning the differential protection of rat liver cells in relation to the absence of this protective effect to HepG2 cells upon exposure to arsenic trioxide (46 fold protection), could indicate that the HepG2 cell line can be killed by much lower concentrations than normal cells (a possible translational advantage).

These findings might be due to the mechanism by which PFT-[alpha] protects cells. It works by temporarily blocking p53 function leading to inactivation of responsive genes and halting p53-mediated apoptosis. Since PFT-[alpha] did not protect both rat liver cells and HepG2 cell line from the cytotoxic effects of cadmium, it could be inferred that the mechanism involving this chemical is p53 independent. In addition, our data also indicated that there was an inherent 8 fold increase in survival for rat liver cells upon exposure to cadmium [45-47].

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Nearly, all chemotherapeutic agents kill cancer cells mainly by p53-induced apoptosis, and PFT-[alpha] works by transient and reversible blockage of p53 function. In the current study, our aim was to explore whether PFT-[alpha], the inhibitor of p53, has any beneficial influence on normal rat liver cells and detrimental effect on HepG2 cell line exposed to genotoxic agents cadmium chloride and arsenic trioxide so that we can extrapolate our findings towards fruitful addition to the already existing data on PFT-[alpha]. The lethally injured and genetically compromised cells are subjected to apoptosis by p53; the guardian of cellular genome [5]. However the mechanism by which cadmium chloride acts as a genotoxic agent is not clear from this study but at least it can be implied that genotoxic affects of cadmium chloride are not p53 dependent [48, 49]. In a different set of experiments, Western blots were used to examine the influence of PFT-[alpha] on transactivation of known p53 responsive genes. These experiments were carried out with rat liver cells and HepG2 cells exposed to arsenic trioxide only since it was apparent from the results of survival assays that cadmium chloride-induced toxicity was probably not p53-mediated. The transcriptional regulation of p53 downstream genes leads to two established consequences, cell cycle arrest and apoptosis, which are at least in part, depend upon p53 activation. While transactivation of target genes by p53 result in their amplification, and appears to be the norm, some genes repression has been described, although this phenomenon was less established in physiological terms [3, 5, 8, and 19].

[FIGURE 3 OMITTED]

Results from Fig 3a show the expression of p53 in rat liver cells in the Western blot analysis. Lane E is the p53 control while lane D shows expression of p53 in control cells (normal rat liver cells unexposed to PFT-[alpha]). Lane C shows no expression of p53 in cells exposed to PFT [alpha] alone (which inhibited p53 and resulted in protection from arsenic toxicity, Fig 1 and2) and Lane B shows p53 protein expression under influence of arsenic trioxide only. Lane A shows expression of p53 under the influence of both arsenic trioxide and PFT-[alpha].

The p53 protein was physically expressed in HepG2 cells, but its functionality could not be ascertained from data presented here. Fig 3b shows expression of p53 in HepG2 cells. Lane E was the p53 control while Lane D shows expression of p53 in unexposed HepG2 cells, which was showing no band. Lane C shows cells exposed to PFT-[alpha] only and these are not expressing p53 compared to normal rat liver cells. Bands in Lanes B and A show the expression in HepG2 cells exposed to arsenic trioxide alone or under the influence of both arsenic trioxide and PFT-[alpha] respectively where both showed significant p53 expression.

Fig 3c shows the expression of PCNA in HepG2 cells. Expression can be seen in all lanes with almost equal intensity showing no difference for the treatment. Lane E is the PCNA control while Lane D shows the expression of the HepG2 cells control. In lane C, exposure to PFT-[alpha] has not affected the basic level of PCNA, and the same is apparent in lane B for arsenic trioxide exposure. Lane D shows a band of lesser intensity for both chemicals. From these findings, it can be assumed that PFT-[alpha] did not have any stimulating or inhibiting affect on PCNA level in HepG2 cells. Despite further repeated attempts, no bands were detected for the expression of PCNA in rat liver cells exposed to arsenic, PFT[alpha] or both. Mdm2 was also not expressed in both cell types in response to both chemicals. Cyclin G, bcl2 and p21 expressions failed to respond to any treatment in both cell types (data not shown).

Table 2 depicts the influence of PFT[alpha] on transactivation of p53 responsive genes and on p53 expression in rat liver and HepG2 cells. PCNA in HepG2 cell line was not influenced by the chemical exposure. The protective effect against p53 expression in rat liver cells has been demonstrated in this study (Fig 2a). In response to arsenic exposure, our data showed transactivation of p53 except for PFT[alpha] in rat liver cells, in contrast, both p53 and PCNA were transactivated in HepG2 cells in response to both arsenic trioxide alone or in combination with PFT-[alpha]. The remaining tested genes (mdm2, bcl2, p21 and cyclin G) were not transactivated in both cell types. The mechanism involved could be because DNA damage activates a set of protein kinases, one of which targets the tumor suppressor protein (p53). The p53 transcription factor is normally maintained at low levels through interaction with the murine double mutant-2 (mdm2) protein that signal its degradation. DNA damage-induced phosphorylation of either p53 or mdm2 prevents the two proteins from interacting, thus stabilizing and activating p53 [3].

The cellular responses to p53 activation are DNA repair, [G.sub.1] growth arrest and apoptosis. The observed results indicated a decrease in apoptosis in rat liver cells in presence of PFT-[alpha] upon exposure to arsenic trioxide. Even though arsenic compounds are not directly genotoxic, it has been reported that arsenic induced apoptosis in vitro is dependent on dose, cell type, test conditions and is mediated by [H.sub.2][O.sub.2], cellular glutathione (GSH), glutathione peroxidase and catalase levels [20]. The p53 protein acts as a tumor suppressor by causing growth arrest or apoptosis in response to DNA damage and other forms of cellular stress [4, 5].

Genotoxic agents up regulate the expressions of mdm2, cyclin G and p21 on induction of p53, while bcl2 and PCNA are repressed [40]. Under the influence of inhibitors of p53, the opposite effects were to be expected. Fig 3 displays expression of p53 in rat liver cells exposed to PFT-[alpha]. Normal rat liver cells are expressing p53 band in Lane D, while cells exposed to PFT[alpha] alone in lane C are not. The explanation for this might be that the expression of wild type p53 has been suppressed by PFT-[alpha]; which is the case, because expression of the p53 protein band is being observed in lanes D in extract of normal cells un exposed to PFT-[alpha]. Interestingly, the expression of p53 band under influence of both arsenic trioxide and PFT-[alpha] in lane A was less prominent than p53 expression in cells exposed to only arsenic trioxide in lane B. This is suggestive of recovery of cells from the toxic stress inflicted by arsenic trioxide and the partial inhibition of p53 expression. These results are encouraging because the total suppression of p53 expression has been demonstrated in normal rat liver cells in lane C and the protection of p53 expression has been observed in lane D (Fig 1 and 2 and table 1). The functional role of these findings will be very interesting to pursue.

Table 2 shows effects of PFT-[alpha] on expression of p53 and p53-responsive genes in response to arsenic trioxide. As expected, the expression of p53 in rat liver cells is inhibited by PFT-[alpha]. In HepG2 cell line p53 expression has been positively influenced. The constitutive expression as well as the transactivation of the PCNA Protein in HepG2 cell line was an impressive finding since both p53 and PCNA was expressed showing another evidence for a mutated p53 in the neoplastic cell line. In attempting to detect the expression of PCNA in rat liver cells exposed to PFT-[alpha], no clear bands in any of the lanes in western blot analysis for samples from these cells could be detected. PCNA possibly has been repressed in all samples by wild type p53, which was expected in normal cells. It is also possible that DNA damage inflicted by arsenic trioxide has been completely abolished by PFT-[alpha].[ 31-37].

Western blots for the detection of mdm2 carried out with protein extracts from both rat liver cells and HepG2 cells exposed to arsenic trioxide in presence of chemical inhibition of p53 by showed no expression of the MDM2 gene. It might be expected that the up-regulation of mdm2 as described in previous studies be hindered, possibly by suppression of p53 by PFT-[alpha], since mdm2 is subject to up-regulation by p53. The role of arsenite in up-regulation of p53 as well as mdm2 should be considered a possibility [19]. Following insult to the genome, inhibition of RB phosphorylation by p53 creates a pause in cell cycle, the cell remains in [G.sub.1] phase allowing time for DNA repair. The control of cell cycle operates through transcriptional up regulation of the CDK inhibitor (CKI), p21. When DNA damage exceeds the capacity of the cell for repair, p53 guides the DNA compromised cell into apoptosis by inducing the expression of the pro-apoptotic protein Bax. In response to up regulation of p53, there is an increase in p21 followed by inhibition of phosphorylation of RB and the cell remains in [G.sub.1] [9-12].

There was an absence of demonstrated induction of p21 in both HepG2 and rat liver cells following exposure to arsenic trioxide in presence of PFT-[alpha]. This might be due to absence of sufficient up-regulation of p53 for the induction of p21. An important gene in connection with human cancer, bcl2 expression is regulated by p53. Shutting off bcl2 facilitates further promotion of apoptosis and elimination of potential cancer cells and providing protection from cancer [12]. When p53 is activated by DNA damage, stress, or oncogenic stimulation, it induces the expression of Bax, throwing off ratio of pro-survival to pro-apoptotic Bcl-2 proteins. An excess of Bax lead to formation of more Bax-Bax homodimers, leading to release of cytochrome C and activation of Apaf -1. Arsenic trioxide normally causes overexpression of bcl2, but in this case, expression of bcl2 in response to arsenic trioxide was generally absent. The possible explanations might be that concentration of arsenic was not optimum or the expression of pro-apoptotic bcl2 was eliminated in these cells [3-8, 30].

In Cyclin G studies utilizing a tetracycline-regulated p53 inducible system in two p53-null tumor cell lines, it was demonstrated that, Cyclin G levels remained unaffected following p53 overexpression, while other p53-inducible genes such as p21 and mdm2 are induced dramatically in a short time. When basal expression levels are high, Cyclin G becomes unresponsive to p53 transcriptional activation [8-10]. It may be possible that Cyclin G mRNA is stabilized in tumor cells and high basal levels of Cyclin G expression may act in a negative feedback loop, suppressing p53-mediated Cyclin G transcription. or, in tumor cells, Cyclin G is being transcribed independent of p53 transcriptional activity at a maximal rate with no further increase in transcription upon p53 induction/activation. Therefore, the constitutive expression of Cyclin G may account for dysregulation of the normal cell cycle in cancer cells, hence affecting their response to various stimuli such as DNA damage. The expressions of cyclin G in HepG2 cell line or in normal rat liver cells exposed to arsenic trioxide in presence of PFT[alpha] were absent. Normally it was expected to be positively induced by p53. The possible cause in this case might not be explained by this study; hence, this area calls for further studies and investigations.

Over all, these results may be promising since they are indicative of differential protection of normal cell in relation to cancer cells with lost functional p53 because of the use of PFT-[alpha] or other compounds with similar properties. It is worth mentioning that many of the chemotherapeutic agents were targeted at the p53-induced apoptotic pathway for destruction of cancer cells and that the derogatory affects of chemotherapeutic agents influence both healthy normal cells and cancerous cells. The biological processes of eliminating harmful elements vary with the p53 protein level in that cell; high levels easing programmed cell death or apoptosis, moderate levels bring growth process to a stand still or growth arrest while low levels oppose both processes and thus lead to cellular proliferation [6-11].

Therefore, a measure that protects normal cells while facilitating removal of DNA-compromised cells is highly desirable. The identification of the stage in p53 pathway that can be targeted by PFT-[alpha] should be achieved by direct overexpression of p53 enhancing the selective targeting of cancerous cells. The reversible and temporary inhibition of p53 function induced by PFT-[alpha], thus hold great prospect for reducing side effects of chemotherapy, materializing its application in the old and infirm for the desired time. It is our expectation that future findings will provide additional information on potential mechanisms involved in toxicity induced by DNA damaging agents and the differential protection of normal tissues by PFT-[alpha].

CONCLUSIONS

Arsenic trioxide is most cytotoxic, p53 mediated, PFT-[alpha] protects normal cells, helps transformed cells in apoptosis suggesting development of clinical applications and novel therapeutic strategies towards alleviating the detrimental and or deadly side affects of anti cancer agents. The gap between array of biochemical properties and interactions ascribed to p53 and physiological consequences that are observed in whole animals may thus be minimized.

From the above findings, we conclude that: (a) Cadmium chloride is more cytotoxic than arsenic trioxide. (b) PFT-[alpha] protects normal cells from the cytotoxic effect of arsenic trioxide but not cancerous cells, a role that could be important in cancer chemotherapy. (c) Arsenic induced cytoxicity in normal cell is p53 dependent. (d) Cadmium chloride induced cytotoxicity in both types of cells is not p53 dependent. Moreover, (e) Transactivation of p53 except for PFT-[alpha] for rat liver cells, in contrast to both p53 and PCNA for HepG2 cell. Gene products mdm2, bcl2, p21 and cyclin G were not transactivated in response to arsenic exposure in both cell types.

ACKNOWLEDGMENTS

We thank Dr. Mark G. Hardy, Interim Dean, College of Science, Engineering and Technology; Dr. Paul B. Tchnouwou, Interim Associate Dean College of Science, Engineering and Technology and Director, Environmental Science Ph.D. Program; Dr. Greg Begonia, Chair, department of Biology Dr Joseph A. Cameron Director Bridges to the Doctorate Program for their cordial support. This research is supported in part by NIGMS R25 GM50117. This work was partially presented at RMBS.

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Ibrahim O. Farah (1*), Rowshan A. Begum (1) and Ali B. Ishaque (2)

(1) Department of Biology, Jackson State University, Jackson, MS 39217 and (2) Department of Natural Sciences, University of Maryland Eastern Shore, Princess Ann, MD 21853.

* Corresponding author: Department of Biology, Box 18540, 1400 Lynch Street, Jackson State University, Jackson, MS 39217. E-mail: [e-mail].

Table 1: Comparative Representation of Toxicity Indices for Arsenic
Trioxide and Cadmium Chloride with and without PFT-[alpha] in Rat Liver
Cells and Neoplastic HepG2 Cell Line.

                                      Treatment of Rat Liver
                       Inhibitor      Cells with toxic agent
Cell survival indices  of p53         Arsenic          Cadmium
Parameters             Treatment      Trioxide         Chloride

[R.sup.2]              PFT-[alpha] +  1.0              0.79
                       PFT-[alpha] -  0.91             0.80
L[C.sub.50][+ or -]SD  PFT-[alpha] +  670[+ or -]8.15  57.72[+ or -]0.8
  ([micro]g/ml)        PFT-[alpha] -  573[+ or -]26.2  58.10[+ or -]5.5

                       Treatment of HepG2
                       Cell Line with toxic agents
Cell survival indices  Arsenic          Cadmium
Parameters             Trioxide         Chloride

[R.sup.2]              0.93             0.93
                       0.97             0.93
L[C.sub.50][+ or -]SD  13.7[+ or -]1.0  6.95[+ or -]2.5
  ([micro]g/ml)        13.4[+ or -]0.6  7.35[+ or -]1.9

[R.sup.2]: Regression co-efficient indicates correlation between
treatment concentration and cell number.
L[C.sub.50] [+ or -] SD (PPM): Concentration of toxic agent that kills
50% of organisms in [micro]g/ml

Table 2: Western blot analysis of the effects of PFT-[alpha] on
transactivation of p53 responsive genes following exposure of normal Rat
Liver Cells and neoplastic HepG2 Cell Line to arsenic trioxide.

                    Treatment of Rat Liver Cells
Parameter                        Arsenic   PFT-[alpha] & Arsenic
Gene product  None  PFT-[alpha]  Trioxide  Trioxide

p53           +     -            +         +
PCNA          -     -            -         -
mdm2          -     -            -         -
Cyclin G      -     -            -         -
p21           -     -            -         -
bcl2          -     -            -         -

                    Treatment of HepG2 Cell Line
Parameter                        Arsenic   PFT-[alpha] & Arsenic
Gene product  None  PFT-[alpha]  Trioxide  Trioxide

p53           -     -            +         +
PCNA          +     +            +         +
mdm2          -     -            -         -
Cyclin G      -     -            -         -
p21           -     -            -         -
bcl2          -     -            -         -

+ = Detection of band indicating expression of gene. - = No band
detected, indicating lack of gene expression. p53 = p53 gene product.
PCNA = Proliferating Cell Nuclear Antigen product. mdm2 = Mouse Double
Minute 2 gene product. Cyclin G = Product of cyclin G gene.