Reduced cellular uptake of cis-dichlorodiammine-platinum by benzaldehyde

Synchronized human NHIK 3025 cells were treated with cis-dichlorodiammineplatinum (cis-DDP) either alone or in combination with benzaldehyde as a 2 h pulse in G1-phase. After this pulse, the cells entered S-phase and the rate of DNA synthesis was measured by DNA-flow cytometric recordings of serial samples. After treatment with 10 microM cis-DDP alone, the rate of DNA synthesis was 38% of the control rate. If 3.2 mM benzaldehyde was present together with 10 microM cis-DDP, the rate of DNA synthesis was 56% of the control rate, this being similar to the rate measured following treatment of cells with 5 microM cis-DDP alone. Thus, the simultaneous presence of benzaldehyde with cis-DDP mitigates the inhibition of DNA synthesis induced by cis-DDP. However, when cells were electropermeabilized during the treatment pulse, benzaldehyde did not protect the cells from cis-DDP-induced cell inactivation. The protective effect of benzaldehyde thus seems to reside with the cell membrane and it seems that benzaldehyde, when present together with cis-DDP, partially inhibits the uptake of cis-DDP into cells. Atomic absorption spectroscopy confirmed that the simultaneous presence of 5 mM benzaldehyde halved the amount of cell-bound platinum from that measured following treatment with cis-DDP alone.     

Evaluation of cisplatin-DNA crosslinks formation with UV-C application by the alkaline Comet-assay

The AIM of the present study was to investigate DNA damage induced by widely applied anticancer preparation cisplatin (cis-DDP) at doses comparable with therapeutic ones in leukocytes of healthy donors. METHODS: DNA damage and repair was estimated by the single cell gel electrophoresis or Comet-assay. For estimation of cis-DDP-induced DNA crosslinks the combined treatment by cis-DDP with the DNA damaging agent UV-C was used. The RESULTS obtained indicate that cis-DDP is forming crosslinks with DNA in human leukocytes and significantly reduce UV-C-induced DNA migration. CONCLUSION: The Comet-assay with UV-C application is a useful tool to detect cis-DDP- induced DNA crosslinks. Data on the cis-DDP-induced DNA damage in vitro may be important for their extrapolation on in vivo level.     

DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.     

In vitro modulating effect of the reversible protein synthesis inhibitor zilascorb (2H) on cis-diamminedichloroplatinum (II)-induced cytotoxicity.

Zilascorb (2H) (5,6-benzylidene-d1-L-ascorbic acid sodium salt), a derivative of deuterated benzaldehyde inducing reversible protein synthesis inhibition, was tested on human NHIK 3025 cells in combination with the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). The inactivating effect of cis-DDP, measured as loss of colony forming ability, was found to increase when a non-toxic treatment with zilascorb (2H) was given simultaneously with, or shortly prior to, treatment with cis-DDP. No potentiating effect was seen when zilascorb (2H) treatment occurred after removal of cis-DDP. Furthermore, the data showed that the potentiating effect of zilascorb (2H) reached a maximum at concentrations of between 2 and 4 mM. The underlying mechanism for this potentiation is unknown, but could possibly be related to the effect of zilascorb (2H) on protein synthesis. Alternatively, it is also possible that generation of free radicals from the ascorbate moiety of the zilascorb (2H) molecule may increase cellular damage induced by cis-DDP, or inhibit repair or restitution of cis-DDP-induced damage. Also, in cells of a cis-DDP-resistant subline, NHIK 3025/DDP zilascorb (2H) was found to potentiate the effect of cis-DDP. The cis-DDP-resistant cells, however, were found to be partly cross-resistant to cytotoxic treatments with zilascorb (2H).     

Effect of H(2)O(2) on cell cycle and survival in DNA mismatch repair-deficient and -proficient cell lines

Patients who develop tumors with Lynch syndrome, which is caused by mutational inactivation of the DNA mismatch repair (MMR) system, have a relatively favorable prognosis compared to patients who develop sporadic tumors. Paradoxically, DNA MMR-deficient cells are resistant to many chemotherapeutic agents, and are capable of bypassing the G2/M checkpoint in vitro. Colon cancers that develop in the setting of Lynch syndrome show an abundant recruitment of immune cells into tumor tissues, which might be expected to increase oxyradical formation, and make the tumor cells more vulnerable to cell death. We examined the chemosensitivity and cell cycle response to oxidative stress in several MMR-deficient (HCT116, SW48, and DLD1) and -proficient (CaCo2, SW480, and HT29) colorectal cancer cell lines. H(2)O(2) induced a G2/M cell cycle arrest in both MMR deficient and proficient cell lines, however MMR-deficient cell lines were more sensitive to H(2)O(2) toxicity, and the response was more prolonged in MMR-deficient cells. Interestingly, human MutL-homologue (hMLH1-)defective HCT116 and hMLH1-restored HCT116+ch3 cell lines responded to H(2)O(2) with the same degree of G2/M arrest. The survival response of HCT116+ch3 was nearly identical to that of hMLH1-defective HCT116+ch2, although better than the response observed in HCT116 cells. In conclusion, greater cellular sensitivity and G2/M arrest in response to oxidative stress in MMR-deficient colorectal cancer cells could be one of the reasons for the more favorable prognosis seen in patients with Lynch syndrome. However, this sensitivity appears not to be a direct result of a deficient MMR function, but is more likely attributable to spectrum of target gene mutations that occurs in MMR-deficient tumors.     

Antagonism between curcumin and the topoisomerase II inhibitor etoposide: A study of DNA damage, cell cycle regulation and death pathways

The use of combinations of chemotherapy and natural products has recently emerged as a new method of cancer therapy, relying on the capacity of certain natural compounds to trigger cell death with low doses of chemotherapeutic agents and few side effects. The current study aims to evaluate the modulatory effects of curcumin (CUR), Nigella sativa (NS) and taurine on etoposide (ETP) cytotoxicity in a panel of cancer cell lines and to identify their underlying mechanisms. CUR alone showed potent antitumor activity, but surprisingly, its interaction with ETP was antagonistic in four out of five cancer cell lines. Neither taurine nor Nigella sativa affect the sensitivity of cancer cells to ETP. Examination of the DNA damage response machinery (DDR) showed that both ETP and CUR elicited DNA double-strand breaks (DSB) and evoked γ-H2AX foci formation at doses as low as 1 μg/ml. Cell cycle analysis revealed S phase arrest after ETP or CUR application, whereas co-treatment with ETP and CUR led to increased arrest of the cell cycle in S phase (MCF-7 cells) or the accumulation of cells in G 2/M phases (HCT116, and HeLa cells). Furthermore, cotreatment with ETP and CUR resulted in modulation of the level of DNA damage induction and repair compared with either agent alone. Electron microscopic examination demonstrated that different modalities of cell death occurred with each treatment. CUR alone induced autophagy, apoptosis and necrosis, whereas ETP alone or in combination with CUR led to apoptosis and necrosis. Conclusions: Cotreatment with ETP and CUR resulted in an antagonistic interaction. This antagonism is related, in part, to the enhanced arrest of tumor cells in both S and G 2/M phases, which prevents the cells from entering M-phase with damaged DNA and, consequently, prevents cell death from occurring. This arrest allows time for the cells to repair DNA damage so that cell cycle -arrested cells can eventually resume cell cycle progression and continue their physiological program.     

Modifying effect of cinnamaldehyde and cinnamaldehyde derivatives on cell inactivation and cellular uptake of cis-diamminedichloroplatinum(II) in human NHIK 3025 cells

The effect of cinnamaldehyde and various cinnamaldehyde derivatives on cell inactivation induced by cis-diamminedichloroplatinum(II) (cis-DDP) was investigated using human NHIK 3025 cells in culture. Cell inactivation was measured as a loss in the ability of single cells to give rise to macroscopic colonies following drug treatment. It was found that cinnamaldehyde and alpha-chlorocinnamaldehyde potentiated the cell-inactivating effect when used simultaneously with cis-DDP without increasing the amount of cell-associated platinum. In contrast, a protective effect with respect to cell inactivation was found when cells were treated with cis-DDP in combination with hydrocinnamaldehyde or alpha-methylcinnamaldehyde. At higher concentrations (greater than 1 mM) all cinnamaldehyde derivatives reduced cellular uptake of cis-DDP. Therefore, while protection from cis-DDP-induced cell inactivation involves reduced platinum uptake, potentiation by cinnamaldehyde and cinnamaldehyde derivatives does not seem to be due to an increase in intracellular platinum. We propose that cinnamaldehyde may compete with cis-DDP in nucleophilic addition reactions involving intracellular sulfhydryls.     

Potentiation of cis-dichlorodiammineplatinum (II)-induced cytotoxicity by methylglyoxal

The dicarbonyl compound methylglyoxal (MG) potentiated the cell inactivating effect of cis-dichlorodiammine-platinum (II) (cis-DDP) when cultured human NHIK 3025 cells were treated with both drugs in simultaneous combination. While a 2 h treatment with cis-DDP alone resulted in a surviving fraction of 0.024 +/- 0.008, the simultaneous presence of 0.5 mM MG, a non-cytotoxic concentration, reduced the fraction of cells surviving treatment to 0.0009 +/- 0.0001. Although the cell inactivating effect of cis-DDP was potentiated, the amount of cell-associated platinum was not affected by MG. Glyoxal, another dicarbonyl compound, did not affect cis-DDP-induced cytotoxicity in any manner, nor did the aliphatic aldehydes acetaldehyde and propionaldehyde. Cell inactivation induced by MG alone at concentrations above 0.5 mM could be prevented by the simultaneous presence of cysteine or glutathione, indicating that cellular sulfhydryls may be involved in the expression of MG-induced cytotoxicity. Furthermore, MG-induced cell inactivation was increased by a dose-modifying factor of 2.6 when NHIK 3025 cells were first pretreated with buthionine sulfoximine, a glutathione synthesis inhibiting compound. We propose that MG reacts with cellular sulfhydryls which may also be involved in the mediation of cis-DDP cytotoxicity. Reactions between cellular sulfhydryls and MG therefore increase the cytotoxicity of cis-DDP by allowing more platinum to react with cellular targets, not by increasing the amount of platinum entering cells.     

Synthetic retinoid CD437 induces cell-dependent cycle arrest by differential regulation of cell cycle associated proteins

BACKGROUND: The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene (CD437) exhibits a wide spectrum antitumor activity through induction of cellular apoptosis and cell cycle arrest. We investigated the effects and mechanisms of CD437 on cell cycle arrest of gastric cancer cells. MATERIALS AND METHODS: The activities of CD437 on cell growth were analyzed by measuring total cellular DNA. The effects of CD437 on cell cycle phase distribution were analyzed using flow cytometry. The levels of cell cycle associated proteins were analyzed by Western blot. The activities of cyclin-dependent kinases (cdks) were analyzed by phosphorylation of the histone H1 protein. RESULTS: CD437 at concentrations between 0.1 and 10 microM profoundly suppressed the growth of all six gastric cancer cell lines. Growth suppression associated with induction of G0/G1 or G2/M arrest was cell-line-dependent. CD437 decreased levels of cdk inhibitor p21 in G2/M-arrested SC-M1 cells. However, CD437 increased p21 levels in G0/G1-arrested AGS cells. Total and activated cyclin-dependent kinases were differentially regulated by CD437 in AGS and SC-M1 cells. CD437 (1 microM) induced activity of cdk2- and p34cdc2-associated H1 kinase by 14.6- and 1.8-fold, respectively, in SC-M1 cells. In contrast, CD437 slightly increased (1.6-fold) the cdk2-associated H1 kinase activity in AGS cells. CONCLUSION: CD437 profoundly suppressed the growth of gastric cancer cells, which was associated with cell-dependent induction of G0/G1 or G2/M arrest. The differential regulation of p21 that leads to alteration in the activity of cdks may play a critical role in cell-line-dependent regulation of cell cycle arrest following treatment with CD437.     

Kinetics and mechanism of the 1-beta-D-arabinofuranosylcytosine-induced potentiation of cis-diamminedichloroplatinum(II) cytotoxicity

Certain aspects of the potentiation induced by 1-beta-D-arabinofuranosylcytosine (ara-C) on cis-diamminedichloroplatinum(II) (cis-DDP) cytotoxicity were investigated. The time dependency of additions of ara-C and cis-DDP was established by allowing cells to grow for various intervals in fresh medium following the removal of one agent before adding the second one. ara-C had no potentiating effect on cis-DDP toxicity when given to the cells before the addition of cis-DDP. When the experiment was reversed so that cis-DDP was added first and ara-C second, a slight potentiating effect was observed even if the drugs were added 4 h apart. The optimal toxic effect was obtained when ara-C and cis-DDP were added together. Continuous exposure of cells to concentrations of ara-C and cis-DDP 10 times lower than those used in pulse treatment experiments resulted in an additive rather than a synergistic effect. ara-C, unable to kill cells in pulse treatment, killed 96% of the cells after 24 h of continuous incubation. Thiourea was able to prevent the cytotoxic effect of cis-DDP in a concentration-dependent manner when given to the cells immediately following their treatment with cis-DDP; at 0.1 M thiourea, the cytotoxic effect of cis-DDP was almost completely prevented. Similar results were obtained when the cells were exposed to a combination of cis-DDP and ara-C. In this case, 0.1 M thiourea resulted in over 80% survival of cells treated with the drug combination. Thiourea had to be added to the cells either together with cis-DDP or immediately following removal of the drug in order to completely prevent the cytotoxic effect. A similar time factor was involved when cells were treated with a combination of cis-DDP and ara-C before their exposure to thiourea, but in this case thiourea was only able to prevent completely the cytotoxic effect when added simultaneously with the drug combination. In other experiments, the effect of thiourea on cis-DDP-induced DNA cross-linking was measured by the alkaline elution technique. Thiourea was capable of preventing DNA cross-link formation both in cells treated with cis-DDP alone, and in cells exposed to the combination of cis-DDP and ara-C These observations further support the contention that ara-C potentiates cis-DDP cytotoxicity by increasing the ability of the platinum compound to form earlier, more stable DNA cross-links regardless of whether it is present in free or monoadducted form.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radiation-induced phosphorylation of Chk1 at S345 is associated with p53-dependent cell cycle arrest pathways

Because DNA damage-inducible cell cycle checkpoints are thought to protect cells from the lethal effects of ionizing radiation, a better understanding of the mechanistic functions of cell cycle regulatory proteins may reveal new molecular targets for cancer therapy. The two major regulatory proteins of G2 arrest are Chk1 and p53. Yet, it is unclear how these two proteins interact and coordinate their functional roles during radiation-induced G2 arrest. To determine Chk1's role in p53-dependent G2 arrest, we used p53 proficient cells and examined expression of G2 arrest proteins under conditions in which G2 arrest was inhibited by the staurosporine analog, UCN-01. We found that UCN-01 inhibited both G1 and G2 arrest in irradiated p53 proficient cells. The arrest inhibition was associated with suppression of radiation-induced expression of both p21 and 14-3-3 sigma -- two known p53-dependent G2 arrest proteins. The suppression occurred despite normal induction of p53 and normal phosphorylation of p53 at S20 and Cdc25C at S216 -- the two known substrates of Chk1 kinase activity. In contrast, we showed that radiation-induced phosphorylation of Chk1 at S345 was associated with binding of Chk1 to p53, p21, and 14-3-3 sigma, and that UCN-01 inhibited S345 phosphorylation. We suggest that DNA damage-induced phosphorylation of Chk1 at S345, and subsequent p53 binding, links Chk1 with p53 downstream responses and may provide a coordinated interaction between DNA damage responses and cell cycle arrest functions.     

X-ray-induced damage repair in exponentially growing and growth arrested confluent poly(adenosine diphosphate-ribose) polymerase-deficient V79 chinese hamster cell line

We studied the role of PARP in X-ray-induced damage repair using V79 Chinese hamster cells and two derivative cell lines ADPRT54 and ADPRT351 deficient in poly(adenosine diphosphate-ribose) polymerase (PARP) activity. We previously demonstrated that these PARP-deficient cells had drastically reduced levels of p53. Further, these cells were also deficient in downstream endpoints of p53 signaling. In the present study we showed that exponentially growing ADPRT54 and ADPRT351 were hypersensitive to X-radiation compared to the parental V79 cells. Under this condition of growth, although the parental V79 cells exhibit G1 arrest in response to X-irradiation, the PARP-deficient cells do not undergo this specific p53-dependent cell cycle arrest. In contrast, all the cell lines showed similar sensitivity to X-radiation under growth arrested conditions. Further, all the cell lines were equally proficient in performing potentially lethal damage repair (PLDR). Our findings suggest that: i) PARP is involved in X-ray-induced damage repair in replicating cells; ii) PARP is not required for X-ray-induced damage repair in quiescent cells; iii) PARP does not participate in PLDR; iv) deficiency of PARP may potentiate the cytotoxicity of X-irradiation by interfering with the p53-dependent G1 block that occurs after X-irradiation. These results suggest the intriguing possibility that the approach of inhibition of PARP combined with X-radiation may have therapeutic potential for the treatment of fast growing tumors. However, this approach may not be beneficial for slow growing/quiescent tumors.     

Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PK(CS) (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by gammaH2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation-induced and etoposide-induced DSB. NU7441 appreciably increased G(2)-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use.     

Differential growth inhibition by 5-fluorouracil in human colorectal carcinoma cell lines

The effects of 5-fluorouracil (5-FU) on cell growth were investigated using a primary culture of human fibroblasts, MRC-5, and three established human colon cancer cell lines, DLD-1, LoVo and SW620. Detailed flow cytometric analyses revealed differential growth inhibition among these cell lines including three modes of cell growth modulation: (a) loss or accumulation of S phase cells; (b) G2/M block; and (c) G1-S arrest. From analyses on the amount of 5-FU incorporated into cellular RNA and the activity of thymidylate synthase (TS), suppression of TS and depletion of dTTP, a possible consequence of the former, was considered to be the major action of 5-FU in these cells. Differences in the cellular responses to the nucleotide pool imbalance appeared to make the cell growth modulation diverse. Loss of S phase cells and G1-S phase arrest were evident in p53 wild-type cells, MRC-5 and LoVo. Cells proficient in DNA mismatch repair, SW620 and MRC-5, showed marked modulations in S-G2/M progression. These findings suggest that multiple factors, including p53 and DNA mismatch repair, participate in diverse cell growth modulations in cells treated with 5-FU. Cellular resistance to 5-FU correlated well with a loss of modulations in S-G2/M progression, rather than with a defect of G1-S arrest, which suggests the significance of DNA mismatch repair as a factor affecting the sensitivity of cells to 5-FU.     

S-Phase arrest by nucleoside analogues and abrogation of survival without cell cycle progression by 7-hydroxystaurosporine

The mechanisms of resistance to nucleoside analogues established in preclinical models are rarely found in primary tumors resistant to therapy with these agents. We tested the hypothesis that cells sense sublethal incorporation of analogues into DNA during replication and react by arresting further DNA synthesis and cell cycle progression. After removal of drug, cells may be able to repair damaged DNA and continue proliferation, thus escaping nucleoside analogue toxicity. As a corollary, we evaluated whether dysregulation of this mechanism causes cell death. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, we found that DNA synthesis decreased, cells arrested in S-phase transit, and 60-70% of the population accumulated in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium. S-phase-arrested cells were then treated with otherwise nontoxic concentrations of UCN-01, which caused rapid onset of apoptosis without cell cycle progression specifically in cells with an S-phase DNA content. Thus, S-phase arrest by nucleoside analogues sensitizes cells to UCN-01, which appears to activate signaling for death mechanisms and/or inhibit survival pathways. These results differ from those in cells arrested at the G2 checkpoint, in which UCN-01 abrogates cell cycle arrest, permitting cells to progress in the cell cycle before apoptosis.     

GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity

In response to DNA damage, the cell cycle checkpoint is an important biological event in maintaining genomic fidelity. Gadd45, a p53-regulated and DNA damage inducible protein, has recently been demonstrated to play a role in the G2-M checkpoint in response to DNA damage. In the current study, we further investigated the biochemical mechanism(s) involved in the GADD45-activated cell cycle G2-M arrest. Using the tetracycline-controlled system (tet-off), we established GADD45-inducible lines in HCT116 (wild-type p53) and Hela (inactivated p53 status) cells. Following inducible expression of the Gadd45 protein, cell growth was strongly suppressed in both HCT116 and Hela cells. Interestingly, HCT116 cells revealed a significant G2-M arrest but Hela cells failed to arrest at the G2-M phases, indicating that the GADD45-activated G2-M arrest requires normal p53 function. The GADD45-induced G2-M arrest was observed independent of p38 kinase activity. Importantly, induction of Gadd45 protein resulted in a reduction of nuclear cyclin B1 protein, whose nuclear localization is critical for the completion of G2-M transition. The reduced nuclear cyclin B1 levels correlated with inhibition of Cdc2/cyclin B1 kinase activity. Additionally, overexpression of cyclin B1 substantially abrogated the GADD45-induced cell growth suppression. Therefore, GADD45 inhibition of Cdc2 kinase activity through alteration of cyclin B1 subcellular localization may be an essential step in the GADD45-induced cell cycle G2-M arrest and growth suppression.     

Alkylator resistance in human B lymphoid cell lines: (1). Melphalan accumulation, cytotoxicity, interstrand-DNA-crosslinks, cell cycle analysis, and glutathione content in the melphalan-sensitive B-lymphocytic cell line (WIL2) and in the melphalan-resistant B-CLL cell line (WSU-CLL)

Two human B lymphoid cell lines WIL2 (melphalan sensitive. ***IC50:8.57 +/- 1.08 mM) and WSU-CLL (melphalan resistant, ***IC50:223.18 +/- 6.45 mM) were used as models to study alkylator resistance in human lymphoid cells. Melphalan transport studies demonstrated decreased initial melphalan accumulation in WSU-CLL cells as compared to WIL2 cells. Lineweaver-Burk plots of the rate of initial melphalan uptake showed an approximately 3.5-fold decrease of Vmax in WSU-CLL cells as compared to WIL2 cells. Melphalan transport was inhibited by 2-amino-bicyclo[2,2,1] heptane-2-carboxylic acid(BCH) in both cell lines, indicating that the amino acid transport (System L, which is sodium independent and inhibited by BCH) is functional in these two cell lines. Only a minor degree of inhibition of melphalan transport was noted after sodium depletion (System ASC, which is sodium dependent and unaffected by BCH). Interstrand-DNA-cross-link formation showed a highly significant correlation with in-vitro cytotoxicity in both two cell lines. However, the melphalan concentration at which such interstrand DNA cross-linking occurred differed significantly when WIL2 cells and WSU-CLL cells were compared. The kinetics of interstrand-DNA-cross-link formation and removal following treatment with melphalan also differed significantly, with WSU-CLL cells, showing a much more rapid rate of removal of interstand DNA cross-links as compared to WIL2 cells. Cell cycle analysis showed that melphalan treatment resulted in the progressive arrest of the WSU cells in G1 and G2 phases. But WIL2 cells failed to enter G1 or G2 arrest after melphalan treatment, suggesting an increased rate of DNA repair occurring in melphalan-resistant WSU-CLL cells. There was no significant difference between the two cell lines in regard to either glutathione content or glutathione-S transferase activity. These findings indicate that multiple factors are associated with alkylator resistance in lymphoid cells including alteration of uptake, DNA repair and cell cycle progression. However no evidence for alteration in glutathione content and glutathione-S-transferase activity was found.     

Inhibition of proteasome-dependent degradation of Wee1 in G2-arrested Hep3B cells by TGF beta 1

Transforming growth factor beta1 (TGF beta 1)-induced G2 arrest was observed when a proliferation inhibitory function of the retinoblastoma protein (Rb) was compromised, but the mechanism underlying the G2 arrest was poorly characterized compared with that of G1 arrest. In the present study, we characterized G2 arrest induced by TGF beta1 (1 ng/mL) in the Rb-negative hepatoma cell line (Hep3B) and compared with G1 arrest in the Rb-positive hepatoma cell line (Huh7). Activities of cyclin-dependent kinases (CDK) 2 and cell division cycle (CDC) 2 were markedly decreased at 24 h, the time when cell-cycle arrest became apparent in both cell lines. However, considerable amounts of inactive CDC2-cyclinB1 complexes were present in the nucleus of G2-arrested Hep3B but were not present in G1-arrested Huh7. The inhibitory phosphorylation of CDC2 on Tyr-15 was significantly elevated at 12-24 h, and its levels gradually declined during G2 arrest in Hep3B. In particular, augmentation of CDK inhibitors p21cip1 and p27kip1 and Wee1 kinase and diminution of CDC25C phosphatase coincided with induced Tyr-15 phosphorylation and inhibition of CDC2. Wee1 in Hep3B was unstable and was degraded in a proteasome-dependent manner, but it became substantially stabilized within 6 h of TGF beta 1 treatment. Moreover, a Wee1 inhibitor, PD0166285, abrogated the TGF beta 1-induced G2 arrest in Hep3B. These findings suggest that TGF beta 1 induced G2 arrest in Hep3B at least in part through stabilization of Wee1 and subsequent increase in Tyr-15 phosphorylation and inhibition of CDC2.     

MDM-2 antagonists induce p53-dependent cell cycle arrest but not cell death in renal cancer cell lines

Renal cell cancers (RCC) are notoriously resistant to chemotherapy and radiotherapy. While mutations of the p53 tumor suppressor gene frequently contribute to therapy resistance in other epithelial cancers, p53 mutations are relatively rare in RCC. To date, there is conflicting evidence as to whether p53 signaling and function are otherwise proficient or defective in tumors with wild-type p53. In this study, we assayed p53 function in a series of RCC cell lines and normal proximal epithelial tubule cells using two different MDM-2 antagonists, Nutlin-3a and MI-219. Most cell lines with wild-type p53 responded to MDM-2 antagonists as evidenced by induction of p53 and its target gene p21. RCC cell lines treated with MDM-2 antagonists consistently accumulated in the G2/M phase of the cell cycle and this event was associated with inhibition of proliferation in RCC cell lines but not in normal proximal epithelial tubule cells. MDM-2 antagonists did not induce significant cell death in RCC cell lines, even with induction of p53-dependent pro-apoptotic genes. In contrast, MDM-2 antagonists caused significant cell death in LNCaP prostate adenocarcinoma cells. RCC cell lines with reduced p53, either by mutation or through ectopic expression of p53 shRNA, demonstrated enhanced sensitivity to cell death following sequential treatment with DNA damage and G2/M checkpoint abrogation. Our results suggest that wild-type p53 RCC cell lines are proficient in p53-dependent cell cycle arrest but defective in p53-dependent cell death.