Modification of cell proliferation with inhibitors.

Recent developments on mechanisms that control cell multiplication, using molecular biology, are renewing interest in inhibitors and activators. A great deal of information has been gained in the past through the use of chemicals that modify passage through the cell cycle. The kinds of inhibitors, their sites of action that disrupt functions essential for proliferation, their usefulness in synchronizing cultures and, importantly, their therapeutic value, have been the subject of many investigations.

Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumor cell lines.

Subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells (NMECs) but not in mammary tumor cell lines (TMECs), led to the cloning of the human gap junction gene connexin 26 (Cx26), identified by its sequence similarity to the rat gene. Two Cx26 transcripts derived from a single gene are expressed in NMECs but neither is expressed in a series of TMECs. Northern analysis using rat Cx probes showed that Cx43 mRNA is also expressed in the normal cells, but not in the tumor lines examined. Connexin genes Cx31.1, Cx32, Cx33, Cx37, and Cx40 are not expressed in either normal cells or the tumor lines examined. In cell-cell communication studies, the normal cells transferred Lucifer yellow, while tumor cells failed to show dye transfer. Both Cx26 and Cx43 proteins were immunolocalized to membrane sites in normal cells but were not found in tumor cells. Further analysis demonstrated that Cx26 is a cell-cycle regulated gene expressed at a moderate level during G1 and S, and strongly up-regulated in late S and G2, as shown with lovastatin-synchronized NMECs. Cx43, on the contrary is constitutively expressed at a uniform low level throughout the cell cycle. Treatment of normal and tumor cells with a series of drugs: 5dB-cAMP, retinoic acid, okadaic acid, estradiol, or TGFb had no connexin-inducing effect in tumor cells. However, PMA induced re-expression of the two Cx26 transcripts but not of Cx43 in several TMECs. Thus Cx26 and Cx43 are both downregulated in tumor cells but respond differentially to some signals. Modulation of gap-junctional activity by drug therapy may have useful clinical applications in cancer.

Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase.

Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. Here we identify the ubiquitin ligase responsible for cyclin E ubiquitination as SCFFbw7 and demonstrate that it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCFFbw7 catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and Drosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation.

Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms.

Cyclin E is a G(1) cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G(1)-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G(2)/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein.

Inhibition of cyclin-dependent kinases by p21.

p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.

Novel splice variants of cyclin E with altered substrate specificity.

Cyclin E, a G(1) cyclin, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In this study we have examined the possibility that the shortened mRNA splice variants could give rise to tumor-specific cyclin E LMW proteins. We used the Splice Capture method to identify, enumerate and isolate known spliced mRNAs and to look for previously undetected mRNA forms of cyclin E that might be translated into the LMW proteins. We show that a new splice variant of cyclin E found in tumor cells isolated by the Splice Capture strategy, named Delta48, activates CDK2 more robustly than full-length cyclin E when assayed from transiently transfected cells with the natural substrate GST-Rb. We also found the Splice Capture method to be superior to the conventional RNase protection assay in analyzing the cyclin E mRNA present in normal and tumor cells. Splice Capture enumerated the relative abundance of known forms of cyclin E mRNA and easily discovered new splice variants in both normal and tumor cells. We conclude that the abundance of cyclin E splice variants in cells may represent a novel form of regulation of cyclin E, and if translated they show altered substrate specificity compared to the full length form of cyclin E.

Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells.

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G(0)/G(1)while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G(0)/G(1)arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.

Activation of the estrogen-signaling pathway by p21(WAF1/CIP1) in estrogen receptor-negative breast cancer cells.

BACKGROUND: Estrogen stimulates the proliferation of cells in normal mammary glands and most estrogen receptor (ER)-positive mammary carcinomas by binding to the ER and promoting the transcription of ER-responsive genes. In cells with functional ERs, estrogen mediates the transition of cells from the G(1) to S phase of the cell cycle. Several cell cycle regulatory proteins have been implicated in the ER-signaling pathway involved in estrogen-mediated growth stimulation and antiestrogen-mediated growth arrest. We sought to determine whether p21, a cyclin-dependent kinase inhibitor, is a component of this pathway and, if so, whether it can mediate estrogen's action in ER-negative breast cancer cells. METHODS: We overexpressed p21 with a tetracycline-inducible system in ER-negative, p21-negative breast cancer cells. Activity of the ER-signaling pathway was monitored in transient transfection assays by using constructs in which the ER promoter or the estrogen-response element (ERE) controls Luciferase expression. The growth-modulating effects of estradiol and antiestrogens on p21-overexpressing clones were assessed. All P: values are from two-sided tests. RESULTS: A strong positive association was found between the expression of p21 and ER in nine breast cancer cell lines and in tumor samples from 60 patients with breast cancer (P:<. 001). Overexpression of p21 in a p21-negative, ER-negative cell line induced both the ER and ERE promoters in an estrogen-responsive manner. Last, stable p21 clones that also lack the expression of wild-type ER were responsive to the growth-inhibitory effects of ICI 182,780, a potent antiestrogen, and the growth-stimulatory effects of 17beta-estradiol. CONCLUSION: The ability of p21 to mediate the activation of the estrogen-signaling pathway in ER-negative tumor cells suggests that p21 plays a novel role in this pathway, a finding that also has important clinical implications.

Protection of normal proliferating cells against chemotherapy by staurosporine-mediated, selective, and reversible G(1) arrest.

BACKGROUND: A major limiting factor in human cancer chemotherapy is toxicity in normal tissues. Our goal was to determine whether normal proliferating cells could be protected from chemotherapeutic agents by taking advantage of the differential drug sensitivity of cell cycle G(1) checkpoint in normal and cancer cells. METHODS: Normal mammary epithelial cells and mammary cancer cells were initially treated with staurosporine at a cytostatic (i.e., nonlethal) concentration, which preferentially arrests normal cells in the G(0)/G(1) phase of the cell cycle without affecting the proliferation of tumor cells. After the selective arrest of normal cells in G(0)/G(1), both normal and tumor cells were treated with doxorubicin or camptothecin, two cytotoxic (i.e., lethal) chemotherapeutic agents. Cells were then allowed to recover in drug-free medium for 12 days. RESULTS: After pretreatment of both normal and tumor cells with staurosporine followed by treatment with doxorubicin or camptothecin, tumor cells were selectively killed by chemotherapeutic agents, whereas normal cells resumed proliferation after the drugs were removed. Pretreatment with staurosporine also protected normal circulating lymphocytes that had been induced to proliferate in vitro with phytohemagglutinin from chemotherapeutic agents. Staurosporine-induced arrest of normal cells in G(0)/G(1) phase was reversible, and arrested cells tolerated doses of camptothecin that were more than 100-fold higher than necessary to eradicate all tumor cells in culture. Staurosporine-mediated G(0)/G(1) arrest targets the retinoblastoma protein (pRb) pathway and was accompanied by a rapid decrease in cyclin-dependent kinase (CDK) 4 protein levels, increased binding of CDK inhibitors p21 and p27 to CDK2, and inhibition of CDK2 activity in normal cells. CONCLUSIONS: Breast cancer cells with defective checkpoints regulated by the pRb pathway can be targeted specifically with chemotherapeutic agents, following staurosporine-mediated, selective and reversible G(0)/G(1) arrest in normal cells.

Folinic acid augmentation of the effects of fluoropyrimidines on murine and human leukemic cells.

The effects of the fluoropyrimidines on leukemic cells of mouse and human origin have been studied in the presence of folinic acid. This reduced folate enhanced the cytotoxicity and the growth inhibitory potency of 5-fluorouracil (5-FUra) and of 5-fluoro-2'-deoxyuridine (FUdR) against all cell lines examined. The human leukemic cell lines used (two T- and two B-cells) were affected by these fluoropyrimidines only at substantially higher concentrations than were found to be inhibitory to mouse L1210 cells; however, the enhancement of the activity of the fluoropyrimidines occurred over the same range of folinic acid concentrations in mouse and human cells. Whereas the total intracellular folate pool increased continuously with every increment of folinic acid added to the medium, the enhancement of the potency of the fluoropyrimidines was limited. Augmentation of the effects of FUdR exceeded that of 5-fluorouracil in the human leukemic cells studied. The cytotoxicity of the fluoropyrimidines (as defined by cloning efficiency) was enhanced to a greater extent than was growth inhibition so that an impressive lethal synergism was noted; for instance, exposure of L1210 cells to nontoxic concentrations of 5-fluorouracil or FUdR in the presence of folinic acid resulted in a 98 or 99.9% cell kill, respectively. In contrast to previous predictions, the fluoropyrimidines were more inhibitory to mouse leukemic cells containing folate pools that were suboptimal for growth than for folate-replete cells. Growth rate experiments showed that cells exposed to moderate concentrations of FUdR were initially inhibited but recovered with time, whereas in cells exposed to both FUdR and folinic acid, the initial growth inhibitory effects were sustained. We conclude that folinic acid stabilizes the effects of the fluoropyrimidines on thymidylate synthase of both mouse and human leukemic cell populations and that this enhancement is reflected in both inhibition of the growth of and the lethality to these cells. We suggest that only doses of the fluoropyrimidines that are capable of initially inhibiting thymidylate synthase to a high degree will be synergistic with excess reduced folates.

Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion.

Cyclin-dependent kinase inhibitors (CKIs) p21, p27, p16, and p15 are an essential and integral part of cell cycle regulation. Studies on the expression of these inhibitors in normal versus tumor human breast cancer cells revealed that although p27 and p16 are expressed at higher levels in tumor cells, p21 and p15 expression were higher in normal cells. Analysis on the expression pattern of these proteins throughout the cell cycle in synchronized cells demonstrated a substantial increase in p21 during the S-phase in normal cells and barely detectable expression of p21 in any phase of the tumor cell cycle. Levels of p15, p16, and p27 remained relatively constant throughout the cell cycle of normal and tumor cells. Synchronization of tumor cells by lovastatin, which arrests cells in G1, resulted in increased levels of p21 and p27 with a concomitant decrease in cyclin-dependent kinase 2-associated kinase activity. Synchronization of cells by double-thymidine block did not result in the induction of p21 or p27. These observations suggest that lovastatin causes a profound cell cycle-independent alteration of CKI expression which is distinct from growth factor deprivation or thymidine block.

Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin.

Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to less than or equal to 4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G1 and not in the G0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [3H]uridine, [3H]leucine, and initial [3H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G1 phase.

Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells.

Identification of the genes that are specifically expressed in tumor cells but not in normal cells (oncogenes), or vice versa (tumor suppressor genes), is important for understanding the molecular basis of cancer. The differential display technique was applied to compare mRNAs from normal and tumor-derived human mammary epithelial cells, cultured under the same conditions. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. They exhibit characteristics of the 3' end of eukaryotic mRNA, as predicted by the method. A complementary DNA fragment seen only in the normal cell was used as a probe to isolate its corresponding complementary DNA clone from a library. Northern analysis confirmed its differential expression. Thus, this method can be used for detecting, cloning, and sequencing of genes that are unique to a host of biological and disease processes.

Processing of cyclin E differs between normal and tumor breast cells.

Cyclin E is a G1 cyclin essential for G1 to S-phase transition of the cell cycle with a profound role in oncogenesis. In tumor cells and tissues, cyclin E is overexpressed and present in its lower molecular weight (LMW) isoforms, and it can be used as a prognosticator for poor patient outcome. In this study, we have examined differences in the processing of cyclin E between normal mammary epithelial and breast cancer cell lines. Five NH2-terminally deleted epitope-tagged (FLAG) cyclin E vectors were constructed spanning the range of LMW forms observed in tumor cells. These constructs were transfected into normal and tumor cells and analyzed for the production of cyclin E-FLAG protein products by Western blot analysis with FLAG and cyclin E antibodies. Our results show that only tumor cells had the machinery to process these cyclin E-FLAG constructs to their LMW forms, whereas normal cells mainly expressed the full-length unprocessed form of each protein. Tumor and normal cells always process the cyclin E-FLAG protein in the same way as endogenously expressed cyclin E. This phenomenon is consistent with all of the cell lines used, regardless of transfection efficiency, time of processing posttransfection, or method of transfection. Furthermore, measurement of FLAG-associated kinase activity in the transfectants revealed that the protein products of the cyclin E-FLAG constructs are 10 times more active in tumor cells than in normal cells. These studies suggest that the LMW forms of cyclin E detected at a much higher level in tumor cells arise from posttranslational action of a protease.

Cyclin E, a potential prognostic marker for breast cancer.

A fundamental cause of cancer is changed properties of genetic material, which may deregulate normal development of the tissue or provide selective growth advantage to the tumor cell. This deregulation of cell proliferation results from altered production of a handful of proteins that play key roles in progression through the eukaryotic cell cycle. Some of these proteins include tumor suppressor genes or oncogenes. However, no one general change or alteration of a critical gene has yet been found in all cancers. Using surgical material obtained from patients with various malignancies, we show that breast cancers and other solid tumors, as well as malignant lymphocytes from patients with lymphatic leukemia, show severe quantitative and qualitative alterations in cyclin E protein production independent of the S-phase fraction of the samples. Hence, these alterations represent a true difference between normal versus tumor tissue. In addition, in breast cancer, the alterations in cyclin E expression become progressively worse with increasing stage and grade of the tumor, suggesting its potential use as a new prognostic marker.

PKCiota promotes ovarian tumor progression through deregulation of cyclin E.

The high frequency of relapse of epithelial ovarian tumors treated with standard chemotherapy has highlighted the necessity to identify targeted therapies that can improve patient outcomes. The dynamic relationship between cyclin E and PKCiota frequent overexpression in high-grade ovarian tumors poses a novel pathway for therapeutic investigation. We hypothesized that a phosphoinositide 3-kinase (PI3K)-dependent signaling pathway activating PKCiota perpetuates cyclin E deregulation during ovarian tumorigenesis. We observed a positive correlation between PKCiota and cyclin E in a panel of 19 ovarian cancer cell lines. Modulation of cyclin E had no effect on PKCiota knockdown/overexpression; however, PKCiota differentially regulated cyclin E expression. In the serous ovarian cancer cells (IGROV and OVCAR-3), shPKCiota decreased proliferation, caused a G1 arrest and significantly prolonged overall survival in xenograft mouse models. In vitro, shPKCiota decreased the ability of IGROV cells to grow under anchorage-independent conditions and form aberrant acini, which was dependent on Ad-cyclin E or Ad-LMW-E expression. Reverse-phase protein array analysis of PKCiota wild-type, catalytic active, dominant-negative protein isoforms strengthened the association between phospho-PKCiota levels and PI3K pathway activation. Inhibitors of PI3K coordinately decreased phospho-PKCiota and cyclin E protein levels. In conclusion, we have identified a PI3K/PKCiota/cyclin E signaling pathway as a therapeutic target during ovarian tumorigenesis.

UCN-01-mediated G1 arrest in normal but not tumor breast cells is pRb-dependent and p53-independent.

In this study we investigated the growth inhibitory effects of UCN-01 in several normal and tumor-derived human breast epithelial cells. We found that while normal mammary epithelial cells w were very sensitive to UCN-01 with an IC(50) of 10nM tumor cells displayed little to no inhibition of growth with any measurable IC(50) at low UCN-01 concentrations (i.e. 0-80 nM). The UCN-01 treated normal cells arrested in G1 phase and displayed decreased expression of most key cell cycle regulators examined, resulting in inhibition of CDK2 activity due to increased binding of p27 to CDK2. Tumor cells on the other hand displayed no change in any cell cycle distribution or expression of cell cycle regulators. Examination of E6- and E7-derived strains of normal cells revealed that pRb and not p53 function is essential for UCN-01-mediated G1 arrest. Lastly, treatment of normal and tumor cells with high doses of UCN-01 (i.e. 300 nM) revealed a necessary role for a functional G1 checkpoint in mediating growth arrest. Normal cells, which have a functional G1 checkpoint, always arrest in G1 even at very high concentrations of UCN-01. Tumor cells on the other hand have a defective G1 checkpoint and only arrest in S phase with high concentrations of UCN-01. The effect of UCN-01 on the cell cycle is thus quite different from staurosporine, a structural analogue of UCN-01, which arrests normal cells in both G1 and G2, while tumor cells arrest only in the G2 phase of the cell cycle. Our results show the different sensitivity to UCN-01 of normal compared to tumor cells is dependent on a functional pRb and a regulated G1 checkpoint.

Deregulation of cyclin E in breast cancer.

Cyclin E, a regulatory subunit of cyclin dependent kinase-2, is thought to be rate limiting for the G1/S transition during the mammalian cell cycle. Previously, we showed severe alterations in cyclin E protein expression in human mammary epithelial cell lines and in surgical material obtained from patients with various malignancies. To understand the functional basis of these alterations we analyse here the regulation of cyclin E in breast cancer cells. We find that while cyclin E protein and its associated kinase activity in normal cells are cell cycle regulated, in tumor cells it remains in an active complex throughout the cell cycle. We also analysed cyclin E for possible deletions which could result in its constitutive function and found two novel truncated variants in its coding region. These variant forms of cyclin E were detected in several normal and tumor cell lines and tissue specimens. However, Western blot analysis indicated that only the multiple isoforms of cyclin E protein were expressed in tumor but not the normal tissue specimen, suggesting post transcriptional regulation of cyclin E. Lastly, in vitro analyses indicated that these truncated variant forms of cyclin E are biochemically active in their ability to phosphorylate histone H1. Collectively these observations suggest the presence of more than one form of cyclin E mRNA in all cells, normal and tumor. Once translated in tumor cells, the protein products of these truncated forms could give rise to a constitutively active form of cyclin E containing complexes.

Lovastatin mediated G1 arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21 and p27, independent of p53.

Previously, we reported that lovastatin, a potent inhibitor of the enzyme HMG CoA reductase also acts as an antimitogenic agent by arresting cells in the G1 phase of the cell cycle resulting in cell cycle-independent alteration of cyclin dependent kinase inhibitors (CKIs). In the present study we have investigated the nature of the CKIs (p21 and p27) alterations resulting in G1 arrest in both normal and tumor breast cell lines by lovastatin. We show that even though lovastatin treatment causes G1 arrest in a wide variety of normal and tumor breast cells irrespective of their p53 or pRb status, the p21 and p27 protein levels are not increased in all cell lines treated suggesting that the increase in p21 and p27 protein expression per se is not necessary for lovastatin mediated G1 arrest. However, the binding of p21 and p27 to CDK2 increases significantly following treatment of cells with lovastatin leading to inhibition of CDK2 activity and a subsequent arrest of cells in G1. The increased CKI binding to CDK2 is achieved by the redistribution of both p21 and p27 from CDK4 to CDK2 complexes subsequent to decreases in CDK4 and cyclin D3 expression following lovastatin treatment. Lastly, we show that lovastatin treatment of 76N-E6 breast cell line with an altered p53 pathway also results in G1 arrest and similar redistribution of CKIs from CDK4 to CDK2 as observed in other breast cell lines examined. These observations suggest that lovastatin induced G1 arrest of breast cell lines is through a p53 independent pathway and is mediated by decreased CDK2 activity through redistribution of CKIs from CDK4 to CDK2.