5-Fluorouracil interferes with paclitaxel cytotoxicity against human solid tumor cells

Paclitaxel, a naturally occurring antimitotic agent, has shown efficacy in the treatment of certain solid tumors, particularly metastatic breast carcinoma and drug-refractory ovarian cancers. Recent studies have demonstrated that paclitaxel, in addition to its effects on microtubules and cell cycle arrest, possesses significant cell-killing activity in solid tumor cells by the induction of apoptosis. However, the mechanism by which paclitaxel leads to cell death and its relationship with paclitaxel-induced mitotic arrest is presently unclear. In this study, we attempted to determine whether pre-arresting tumor cells at other phases of the cell cycle could affect paclitaxel-induced apoptosis. We found that 5-fluorouracil (5-FU), another antineoplastic agent that usually arrests tumor cells at the G1-S phase of the cell cycle, could significantly repress the cell-killing activity of paclitaxel in solid tumor cells, even when it was added simultaneously with paclitaxel. Further studies indicated that 5-FU actually inhibits the cytotoxic effects of paclitaxel on both mitotic arrest and apoptotic cell death, suggesting that 5-FU might interfere with paclitaxel cytotoxicity at an early stage, probably by preventing tumor cells from entering G2-M phase. Because recent clinical trials have used a combination of paclitaxel and 5-FU in the treatment of metastatic breast cancers, our results also suggest that the combination of these two drugs might not be as valuable in clinical chemotherapy.     

Enforced CDK4 expression in a hematopoietic cell line confers resistance to the G1 arrest induced by ionizing radiation

In hematopoietic cells, gamma-irradiation causes a p53-dependent transient G1 phase cell cycle arrest. Various extracellular growth inhibitory signals elicit G1 arrest by targeting CDK4. Here we show that in a myeloid cell line, 32D cl 3, enforced expression of CDK4, but not cyclins D2 nor D3, overrides the gamma-irradiation-induced G1 arrest. CDK4 does not confer resistance to the radiation-induced G2 block observed in parental cells. Ectopic expression of CDK4 overcomes the ionizing radiation-induced inhibition of CDK4 and CDK2 kinase activity. The levels of CDK4 protein do not change after exposure to ionizing radiation in either parental cells or those overexpressing CDK4. Ionizing radiation induces the expression of both p53 and p21, and in cells constitutively synthesizing exogenous CDK4, the return of p53 protein levels to baseline is prolonged. Increased levels of p21 are found associated with CDK4, and not CDK2, in the lines overexpressing CDK4, compared to the parental line, after exposure to ionizing radiation. Enforced expression of CDK4 may therefore overcome a gamma-irradiation-induced G1 arrest through the titration of the CDK inhibitor p21 allowing both CDK4 and CDK2 to remain active.     

Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins

There is an increasing interest in identifying potent cancer preventive and therapeutic agents against breast cancer. Silymarin, a flavonoid antioxidant isolated from milk thistle, exerts exceptionally high to complete anticarcinogenic effects in tumorigenesis models of epithelial origin. In this study, we investigated the anticarcinogenic effect of silymarin and associated molecular mechanisms, using human breast carcinoma cells MDA-MB 468. Silymarin treatment resulted in a significantly high to complete inhibition of both anchorage-dependent and anchorage-independent cell growth in a dose- and time-dependent manner. The inhibitory effects of silymarin on cell growth and proliferation were associated with a G1 arrest in cell cycle progression concomitant with an induction of up to 19-fold in the protein expression of cyclin-dependent kinase (CDK) inhibitor Cip1/p21. Following silymarin treatment of cells, an incremental binding of Cip1/p21 with CDK2 and CDK6 paralleled a significant decrease in CDK2-, CDK6-, cyclin D1-, and cyclin E-associated kinase activity with no change in CDK2 and CDK6 expression but a decrease in G1 cyclins D1 and E. Taken together, these results suggest that silymarin may exert a strong anticarcinogenic effect against breast cancer and that this effect possibly involves an induction of Cip1/p21 by silymarin, which inhibits the threshold kinase activities of CDKs and associated cyclins, leading to a G1 arrest in cell cycle progression.     

Socioeconomic geographical links to human immunodeficiency virus seroprevalence among childbearing women in Montreal, 1989-1993

Anti-idiotype (anti-Id) antibody can induce tumor dormancy in a murine B lymphoma, BCL1, by its ability to induce cell cycle arrest and apoptosis (negative signaling). In human B lymphoma, there is accumulating evidence that the antitumor effect of anti-Id or several other B cell-reactive antibodies relates to their ability to act as agonists rather than conventional effector antibodies. In this study, we sought to elucidate the role of cyclins, cyclin-dependent kinases (CDKs), and their inhibitors in anti-IgM-induced cell cycle arrest to better understand the mechanisms underlying cancer dormancy. To accomplish this, we have performed in vitro studies with a human lymphoma cell line (Daudi) because its response to anti-Id (or anti-IgM) is similar to that of a BCL1 cell line, more reagents are available, and the results would be particularly pertinent to therapy of human B cell lymphomas. Our results show that cross-linking of membrane IgM on Daudi cells induces an arrest late in G1 and prevents pRb from becoming phosphorylated. The G1 arrest is correlated with an induction of the CDK inhibitor p21 and reduced CDK2 activity, although the level of CDK2 protein was not changed. Coprecipitation of CDK2 with p21 in anti-IgM-treated cells and the unchanged level of cyclin E suggest that p21 is responsible for the reduction of CDK2 activity and therefore blockade of the cell cycle. The induction of p21 was not accompanied by changes in p53 levels. As a result of the G1 block, cyclin A levels sharply declined by 24 h after anti-IgM treatment. There was no evidence for involvement of CDK4 or CDK6 in the blockade. These results provide evidence that membrane IgM cross-linking on Daudi cells induces expression of p21 and a subsequent inhibition of the cyclin E-CDK2 kinase complex resulting in a block to pRb phosphorylation and cell cycle arrest late in G1.     

Defective expression of the DNA mismatch repair protein, MLH1, alters G2-M cell cycle checkpoint arrest following ionizing radiation

A role for the Mut L homologue-1 (MLH1) protein, a necessary component of DNA mismatch repair (MMR), in G2-M cell cycle checkpoint arrest after 6-thioguanine (6-TG) exposure was suggested previously. A potential role for MLH1 in G1 arrest and/or G1-S transition after damage was, however, not discounted. We report that MLH1-deficient human colon carcinoma (HCT116) cells showed decreased survival and a concomitant deficiency in G2-M cell cycle checkpoint arrest after ionizing radiation (IR) compared with genetically matched, MMR-corrected human colon carcinoma (HCT116 3-6) cells. Similar responses were noted between murine MLH1 knockout compared to wild-type primary embryonic fibroblasts. MMR-deficient HCT116 cells or embryonic fibroblasts from MLH1 knockout mice also demonstrated classic DNA damage tolerance responses after 6-TG exposure. Interestingly, an enhanced p53 protein induction response was observed in HCT116 3-6 (MLH1+) compared with HCT116 (MLH1-) cells after IR or 6-TG. Retroviral vector-mediated expression of the E6 protein did not, however, affect the enhanced G2-M cell cycle arrest observed in HCT116 3-6 compared with MLH1-deficient HCT116 cells. A role for MLH1 in G2-M cell cycle checkpoint control, without alteration in G1, after IR was also suggested by similar S-phase progression between irradiated MLH1-deficient and MLH1-proficient human or murine cells. Introduction of a nocodazole-induced G2-M block, which corrected the MLH1-mediated G2-M arrest deficiency in HCT116 cells, clearly demonstrated that HCT116 and HCT116 3-6 cells did not differ in G1 arrest or G1-S cell cycle transition after IR. Thus, our data indicate that MLH1 does not play a major role in G1 cell cycle transition or arrest. We also show that human MLH1 and MSH2 steady-state protein levels did not vary with damage or cell cycle changes caused by IR or 6-TG. MLH1-mediated G2-M cell cycle delay (caused by either MMR proofreading of DNA lesions or by a direct function of the MLH1 protein in cell cycle arrest) may be important for DNA damage detection and repair prior to chromosome segregation to eliminate carcinogenic lesions (possibly brought on by misrepair) in daughter cells.     

华蟾素注射液对人肝癌HepG-2细胞增殖及周期的影响

Objective: The aim of our study was to investigate the effect of Cinobufacini injection on the proliferation and cell cycle of human hepatoma HepG-2 cells. Methods: Cell proliferation was assessed by MTT assay, cell cycle distributionwas detected by the flow cytometry (FCM). The expression of Cyclin A, CDK2 mRNA levels were examined by RT-PCR.Quantitative colorimetric assay was used to analyze Cyclin NCDK2 activity in HepG-2 cells. Results: Cinobufacini injection significantly inhibited HepG-2 cells proliferation in dose- and time-dependent ways; FCM analysis showed Cinobufacini injection induced cell cycle arrest at S phase; RT-PCR assay showed Cinobufacini injection down-regulated Cyclin A, CDK2expression at mRNA levels; Quantitative colorimetric assay showed Cinobufacini injection deceased Cyclin A/CDK2 activity in HepG-2 cells. Conclusion: Cinobufacini injection can inhibit human hepatoma HepG-2 cells growth, induce cell apoptosis and induce cell cycle arrest at S phase, the mechanism of which might be partly related to the down-regulation of Cyclin A,CDK2 mRNA expression and inhibition of Cyclin A/CDK2 activity.     

The control of protein release from poly(DL-lactide co-glycolide) microparticles by variation of the external aqueous phase surfactant in the water-in oil-in water method

A unique feature of p21 that distinguishes it from the other cyclin-dependent kinase (CDK) inhibitors is its ability to associate with the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerases delta and epsilon. While it is now well established that inhibition of cyclin/CDK complexes by p21 can result in G1 cell cycle arrest, the consequences of p21/PCNA interaction on cell cycle progression have not yet been determined. Here, we show, using a tetracycline-regulated system, that expression of wild-type p21 in p53-deficient DLD1 human colon cancer cells inhibits DNA synthesis and causes G1 and G2 cell cycle arrest. Similar effects are observed in cells expressing p21CDK-, a mutant impaired in the interaction with CDKs, but not in cells expressing p21PCNA-, a mutant deficient for the interaction with PCNA. Analysis of cells treated with a p21-derived PCNA-binding peptide provides additional evidence that the growth inhibitory effects of p21 and p21CDK result from their ability to bind to PCNA. Our results suggest that p21 might inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes, and inhibition of PCNA function resulting in both G1 and G2 arrest.     

Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line

It has been shown recently that expression of p21 is enhanced by paclitaxel. This cytotoxic compound induces mitotic spindle damage resulting in blockade of the mitotic cell cycle associated or not with apoptotic cell death. In the present study, we showed that, in MCF-7 cells, paclitaxel induced accumulation of p21 in cells with a G2/M DNA content, corresponding to cells either in abnormal mitosis or in an interphase-like state (decondensed chromatin) with multiple nuclei. In MCF-7 cells, the increase in p21 was subsequent to the mitotic arrest and was associated with the exit from abnormal mitosis leading to formation of cells with micronuclei. In this cell line, we noted a relationship between the elevation of p21 expression and the inhibition of p34cdc2 activity. High levels of p21 protein were also found to be associated with inactive p34cdc2/cyclin B protein complex after treatment with paclitaxel. Treatment with p21 antisense oligonucleotide partially blocked induction of p21 expression by paclitaxel and significantly reduced survival of MCF-7 cells exposed to this agent. In NIH-OVCAR-3 cells, which are deficient in basal and paclitaxel-induced p21 expression, paclitaxel led to a prolonged activation of p34cdc2 and a delayed mitotic exit associated with apoptotic cell death. These observations suggest that p21 is not required for the mitotic arrest in response to paclitaxel, but argue in favor of a role for this inhibitor in facilitating the exit from abnormal mitosis. This effectively enhances cell survival after paclitaxel-induced spindle damage.     

Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis

Phosphorylation of Bcl-2 protein is a post-translational modification of unclear functional consequences. We studied the correlation between Bcl-2 phosphorylation, mitotic arrest, and apoptosis induced by the anti-tubulin agent paclitaxel. Continuous exposure of human cervical carcinoma HeLa cells to 50 ng/ml paclitaxel resulted in mitotic arrest with a symmetrical bell-shaped curve over time. The number of mitotic cells was highest at 24 h (82%), then declined as arrested cells progressed into apoptosis, and barely no mitotic cells were present at 48-60 h. The time curves of paclitaxel-induced cyclin B1 accumulation and stimulation of Cdc2/cyclin B1 kinase activity were identical and superimposable to that of M phase arrest. In contrast, apoptosis was first detected at 12 h and steadily increased thereafter until the termination of the experiments at 48-60 h, when about 80-96% of cells were apoptotic. Bcl-2 phosphorylation was closely associated in time with M phase arrest, accumulation of cyclin B1, and activation of Cdc2/cyclin B1 kinase, but not with apoptosis. At 24 h, when about 82% of the cells were in mitosis, almost all Bcl-2 protein was phosphorylated, whereas at 48 h, when 70-90% of the cells were apoptotic, all Bcl-2 protein was unphosphorylated. Similar results were obtained with SKOV3 cells, indicating that the association of paclitaxel-induced M phase arrest and Bcl-2 phosphorylation is not restricted to HeLa cells. We used short exposure to nocodazole and double thymidine to synchronize HeLa cells and investigate the association of Bcl-2 phosphorylation with mitosis. These studies demonstrated that Bcl-2 phosphorylation occurs in tight association with the number of mitotic cells in experimental conditions that do not lead to apoptosis. However, a continuous exposure to nocodazole resulted in a pattern of Bcl-2 phosphorylation, M phase arrest, and apoptosis similar to that observed with paclitaxel. The phosphatase inhibitor okadaic acid was found to inhibit the dephosphorylation of phosphorylated Bcl-2 and to delay the progression of nocodazole M phase-arrested cells into interphase. In contrast, the serine/threonine kinase inhibitor staurosporine, but not the tyrosine kinase inhibitor genistein, led to rapid dephosphorylation of phosphorylated Bcl-2 and accelerated the progression of nocodazole M phase-arrested cells into interphase. Immune complex kinase assays in cell-free systems demonstrated that Bcl-2 protein can be a substrate of Cdc2/cyclin B1 kinase isolated from paclitaxel-treated cells arrested in M phase. Taken together, these studies suggest that Bcl-2 phosphorylation is tightly associated with mitotic arrest and fail to demonstrate that it is a determinant of progression into apoptosis after mitotic arrest induced by anti-tubulin agents.     

Mechanisms of cyclin-dependent kinase inactivation by progestins

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of approximately 120 and approximately 200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.     

TGF-beta 1 induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells

TGF-beta1 inhibits the cell cycle progression of many types of cells by arresting them in the G1 phase. This cell cycle arrest has been attributed to the regulatory effects of TGF-beta1 on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of proteins, such as p15INK4b, p21WAF1/Cip1, and p27Kip1, that physically associate with cyclins, cyclin-dependent kinases (Cdk), or cyclin-Cdk complexes. In epithelial cell lines, TGF-beta1 was previously shown to inhibit cell cycle progression through down-regulation of Cdk4 and/or up-regulation of p15INK4b and/or p21WAF1/Cip1. However, TGF-beta1 had little or no effect on the p27Kip1 mRNA and protein levels. In this report, we show that, in contrast to observations in epithelial cell lines, TGF-beta1 increased the p27Kip1 mRNA and protein levels in the murine B cell lines CH31 and WEHI231. This TGF-beta1-mediated induction of p27Kip1 also resulted in an increased association of p27Kip1 with Cdk2 and a decreased Cdk2 kinase activity. In contrast to epithelial cells, however, TGF-beta1 had little or no effect on the Cdk4 and p21WAF1/Cip1 protein levels in these B cells. Finally, although several studies suggested a direct role of p53 in TGF-beta1-mediated cell cycle arrest in epithelial cells, TGF-beta1 inhibited cell cycle progression in CH31 even in the absence of wild-type p53. Taken together, these results suggest that TGF-beta1 induces G1 arrest in B cells primarily through a p53-independent up-regulation of p27Kip1 protein.     

Cell cycle regulation by anticancer agent

Some anticancer agents induce cell cycle arrest. We analyzed the effect of anticancer agents on cell-cycle regulators, such as CDK. Our data suggested that arresting cells in the G2-phase of the cell cycle by cisplatin might be regulated by dephosphorylation of cdc2 kinase. Butyrolactone I inhibits both cdc2 and CDK2 kinase in the cell-free system. The cytotoxic effect of paclitaxel shows mainly in the M-phase of the cell cycle. Suramin inhibits cdc2 kinase. UCN-01, a protein kinase-C inhibitor, also inhibits both cdc2 and CDK2 kinase. Some anticancer agents induce apoptosis.     

Effects of guanine nucleotide depletion on cell cycle progression in human T lymphocytes

Depletion of guanine nucleotide pools after inhibition of inosine monophosphate dehydrogenase (IMPDH) potently inhibits DNA synthesis by arresting cells in G1 and has been shown to induce the differentiation of cultured myeloid and erythroid cell lines, as well as chronic granulocytic leukemic cells after blast transformation. Inhibitors of IMPDH are also highly effective as immunosuppressive agents. The mechanism underlying these pleiotropic effects of depletion of guanine nucleotides is unknown. We have examined the effects of mycophenolic acid (MPA), a potent IMPDH inhibitor, on the cell cycle progression of activated normal human T lymphocytes. MPA treatment resulted in the inhibition of pRb phosphorylation and cell entry into S phase. The expression of cyclin D3, a major component of the cyclin-dependent kinase (CDK) activity required for pRb phosphorylation, was completely abrogated by MPA treatment of T cells activated by interleukin-2 (IL-2) and leucoagglutinin (PHA-L), whereas the expression of cyclin D2, CDK6, and CDK4 was more mildly attenuated. The direct kinase activity of a complex immunoprecipitated with anti-CDK6 antibody was also inhibited. In addition, MPA prevented the IL-2-induced elimination of p27(Kip1), a CDK inhibitor, and resulted in the retention of high levels of p27(Kip1) in IL-2/PHA-L-treated T cells bound to CDK2. These results indicate that inhibition of the de novo synthesis of guanine nucleotides blocks the transition of normal peripheral blood T lymphocytes from G0 to S phase in early- to mid-G1 and that this cell cycle arrest results from inhibition of the induction of cyclin D/CDK6 kinase and the elimination of p27(Kip1) inhibitory activity.     

Inhibition of G1 cyclin expression in normal rat kidney cells by inostamycin, a phosphatidylinositol synthesis inhibitor

We previously reported that inostamycin, an inhibitor of CDP-DG: inositol transferase, inhibited cell proliferation in normal rat kidney (NRK) cells by blocking cell cycle progression at the G1 phase. In the present paper, we report the effect of inostamycin on the serum-induced activation of Ser/Thr protein kinases that are involved in G1 progression. In quiescent NRK cells mitogen-activated protein kinase (MAP kinase) and casein kinase II were activated within 15 min after serum addition. Neither activation was affected by the treatment with inostamycin. However, in the inostamycin-treated cell, cyclin-dependent kinase 2 (CDK2) failed to be activated after serum stimulation. Since serum-induced expression of cyclin E was also suppressed by inostamycin, this inhibitor would appear to block CDK2 activation by inhibiting cyclin E expression. Furthermore, inostamycin also inhibited cyclin D1 expression induced by serum; and consequently, hyperphosphorylation of retinoblastoma protein (pRB) by RB-kinases such as CDK4 and CDK2 was abolished, which would result in elimination of functional inactivation of pRB. Thus, early G1 arrest in NRK cells by inostamycin is due to the inhibition of cyclin D1 and E expressions.     

Cell cycle-dependent tumor necrosis factor apoptosis

To determine if tumor necrosis factor (TNF)-mediated apoptosis affects cells at defined stages of the cell cycle, WEHI-164/2F (WEHI) cells were synchronized at G0-G1 after 3-day cultures in medium containing RPMI 1640 and 0.5% FCS (RPMI-0.5% FCS). The arrested WEHI cells (60-75% in G0-G1) showed increased sensitivity to TNF killing, measured as 48-h 3-(5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays, and 15-h apoptosis by propidium iodide staining and flow cytometry analysis. The TNF killing kinetics of G0-G1-arrested cells was similar to controls, and TNF did not accelerate or retard cell cycle progression of the arrested cells after feeding with fresh RPMI-0.5% FCS. However, TNF inhibited WEHI DNA synthesis as early as 1 h after treatment, and inhibition was proportionate to sensitivity to TNF-induced apoptosis. WEHI cells treated with TNF showed a higher percentage of cells in S phase with concomitant decrease in G0-G1 and G2-M. When cultured for 3-18 h in fresh RPMI-0.5% FCS to allow progression of the G0-G1-arrested cells toward the G1-S boundary, WEHI cells became more sensitive to TNF killing, especially at the 3-9 h time points. Moreover, TNF did not degrade [125I]5-iodo-2'-deoxyuridine-labeled WEHI DNA if the labeled cells were precultured for 9 h in fresh RPMI-0.5% FCS to allow them to pass S phase before the addition of TNF. These results show that TNF-induced apoptosis of WEHI cells is connected to cell cycle events; WEHI targets receive the TNF cytotoxic signal mainly at the G1-S boundary and begin to die by apoptosis as they exit from S phase.     

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 --> S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.     

The Bcr-Abl tyrosine kinase activates mitogenic signaling pathways and stimulates G1-to-S phase transition in hematopoietic cells

Bcr-Abl is a constitutively active tyrosine kinase that is expressed in Philadelphia chromosome (Ph1)-positive human leukemias. Bcr-Abl has been shown to inhibit apoptosis and cause anchorage independent growth. However, its ability to activate mitogenic signaling pathways is controversial. Here we show that Bcr-Abl signaling prevents down-regulation of cyclin-dependent kinase activity and cell cycle arrest after growth factor deprivation of hematopoietic progenitor cells. Using an inducible system to regulate Bcr-Abl expression, we also demonstrate that Bcr-Abl expression is sufficient to induce G1-to-S phase transition, DNA synthesis, and activation of cyclin-dependent kinases in cells that were arrested in G0 by growth factor deprivation. Furthermore, Bcr-Abl activates Ras, Erk, and Jnk pathways as a primary consequence of expression. These data show that Bcr-Abl is one of a select group of oncogenes that is capable of both inhibiting apoptosis and deregulating cell proliferation. The combination of these activities is likely to be important for the progression of CML.     

CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation

The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0.5 microM in vitro. Inhibition was competitive with respect to ATP (Ki = 95 nM), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8.5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1.25 to 20 microM. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation.     

Schedule-dependent interaction of doxorubicin, paclitaxel and gemcitabine in human breast cancer cell lines

We showed previously that a sequential treatment with doxorubicin (4 hr) followed by paclitaxel (24 hr) (Dox-->Pacl) induces a synergistic cytotoxic effect in the BRC-230 breast cancer cell line and in human primary breast cancer cultures. The validity of this experimental finding was confirmed in a clinical phase I/II study on advanced breast cancer patients. To improve the cytotoxic effect obtained by the Dox-->Pacl sequence, we analyzed the effect of adding gemcitabine (Gem) to the Dox-->Pacl sequence in a preclinical study. Our study was performed on BRC-230 and MCF-7 cell lines, and cytotoxic activity was evaluated by the sulforhodamine B assay and the type of drug interaction by Drewinko's test. When Gem (0.01 microg/ml for 24 hr) was given immediately or 24 hr after Dox-->Pacl, an antagonistic cytotoxic effect was observed. Conversely, a synergistic effect was found when Gem was given 48 hr after Dox-->Pacl. From results of flow cytometric analysis, the synergistic effect was attributed to cell cycle perturbation. Cells were arrested in G2-M (95% in treated vs. 21% in control samples) 24 hr after Dox-->Pacl treatment. The block progressively recovered thereafter, and after a further 24 hr, at the time of Gem treatment, the cells progressed into the G1-S phase boundary (the cell cycle phase susceptible to the cytocidal effect of the drug). Our findings suggest that the interactions of Dox, Pacl and Gem are highly schedule- and time-dependent and should be taken into consideration in the planning of clinical protocols.