Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.     

7-Hydroxystaurosporine (UCN-01) induces apoptosis in human colon carcinoma and leukemia cells independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Recombinant PML adenovirus suppresses growth and tumorigenicity of human breast cancer cells by inducing G1 cell cycle arrest and apoptosis

Our previous studies demonstrated that the promyelocytic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing effect in breast cancer cells were examined. We showed that Ad-PML effectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post-infection and a detectable level remained at day 16. Ad-PML significantly suppressed the growth rate, clonogenicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the effect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a significant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphorylated 48 h post-infection. These findings indicate that PML exerts its growth suppressing effects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppressing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy.     

Ornithine decarboxylase over-expression stimulates mitogen-activated protein kinase and anchorage-independent growth of human breast epithelial cells

Inactivation of one X chromosome (X inactivation) in female mammals results in dosage compensation of X-chromosomally encoded genes between sexes. In the embryo proper of most mammals X inactivation is thought to occur at random with respect to the parental origin of the X chromosome. We determined on the cellular level the expression of the X-chromosomally encoded protein dystrophin in skeletal and cardiac muscle of female mice heterozygous for a null mutation of the dystrophin gene (mdx/+). In all muscles investigated (cardiac, anterior venter of digastric muscle, biceps brachii and tibialis anterior muscle) we found a mosaic expression of dystrophin-expressing versus non-expressing cells and determined their proportion with respect to the parental origin of the X chromosome. In all groups of mdx/+ mice the level and pattern of dystrophin expression were found to be dependent on the parental origin of the mdx mutation. Additionally, the extent of dystrophin expression was clearly dependent on the mouse strains (C57BL/10 and BALB/c) used to produce heterozygous mdx/+ mice. Variable differences and patterns of dystrophin expression in skeletal versus cardiac muscle were found that were strictly dependent on the parental source of the mdx mutation and the strain used to breed mdx/+ mice. Moreover, dystrophin expression was found to be different between the right side and the left side of the body in individual muscles, and this difference was clearly dependent on the parental origin of the X chromosome. Our data provide evidence that in the mouse embryo proper there is a non-random distribution of cells showing inactivation of the paternal versus the maternal X chromosome in skeletal and cardiac muscle, indicating a non-random X-inactivation. Besides gametic imprinting, strain-, tissue and position-dependent factors also appear to bias X inactivation.     

Human bone cells stimulate the growth of human breast carcinoma cells

Human breast cancer cells were cultured together with their metastatic target, bone tissue, to analyze possible growth promotion effects. The coculture of human osteosarcoma cells (TE-85) with human mammary carcinoma cells (ZR-75.1) resulted in up to 8.4-fold stimulation of proliferation of the breast tumor cells. Cell contact of the two cultures was permitted through the channels of Nuclepore filters. However, physical contact turned out not to be necessary, since the proliferative stimulus was also mediated by a bone-derived diffusible factor. Conditioned medium (CM), collected from human primary bone cultures, enhanced the rate of proliferation of several breast tissue cell lines (ZR-75.1, BT-20, HBL-100), while some lines were not affected by osteoblast CM. Breast tissue lines responding to bone CM express low to intermediate levels of the c-erbB-2 gene, in contrast to nonstimulated lines, which overexpress the gene. Recent observations of metastatic spread in breast cancer patients suggest a distinctive pattern of secondary tumor distribution in association with c-erbB-2 protein expression. Bone tissue seems to be a preferential target for metastases of c-erbB-2-negative breast tumors.     

Blockade of transforming growth factor-beta signaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells

We have studied the role of autocrine transforming growth factor-beta (TGF-beta) signaling on antiestrogen-mediated growth inhibition of hormone-dependent T47D and MCF-7 human breast carcinoma cells. Tamoxifen treatment increased the secretion of TGF-beta activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-beta receptors in both cell lines. Anti-pan-TGF-beta antibodies did not block anti-estrogen-induced recruitment in G1 and inhibition of anchorage-dependent and -independent growth of both cell lines. Early passage MCF-7 cells, which exhibit detectable type II TGF-beta receptors at the cell surface and exquisite sensitivity to exogenous TGF-beta1, were transfected with a tetracycline-controllable dominant-negative TGF-betaRII (DeltaRII) construct. Although the TGF-beta1 response was blocked by removal of tetracycline in MCF-7/DeltaRII cells, tamoxifen-mediated suppression of Rb phosphorylation, recruitment in G1, and inhibition of cell proliferation were identical in the presence and absence of tetracycline. TGF-beta1 treatment up-regulated the Cdk inhibitor p21 and induced its association with Cdk2 in MCF-7 cells; these responses were blocked by the DeltaRII transgene product. In MCF-7 cells with a functional TGF-beta signaling pathway, tamoxifen did not up-regulate p21 nor did it induce association of p21 with Cdk2, suggesting alternative mechanisms for antiestrogen-mediated cytostasis. Finally, transfection of late-passage, TGF-beta1 unresponsive MCF-7 cells with high levels of TGF-betaRII restored TGF-beta1-induced growth inhibition but did not enhance tamoxifen response in culture. Taken together these data strongly argue against any role for TGF-beta signaling on tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.     

Enhanced growth inhibition and differentiation of fluorodeoxyuridine-treated human colon carcinoma cells by phenylbutyrate

The effect of phenylbutyrate (PB), a nontoxic differentiation inducer, in human colon carcinoma cell lines treated with 5-fluorodeoxyuridine (FUdR) was evaluated. Two HT-29 human colon carcinoma subclones (U4 well differentiated and U9 poorly differentiated) were equally growth inhibited by 16 h of FUdR (0.2 microM) treatment but recovered cell growth in 3-6 days after the removal of FUdR. PB as a single agent had minimal effect on cell growth, but after FUdR treatment, PB inhibited cell growth for 12 days. The inhibition of cell growth in FUdR-treated cells by PB was more sustained in U4 than U9 cells and was associated with an increased and sustained expression of p21waf1 protein, secretion of transforming growth factor beta1, mediators of p53-dependent or -independent G1 cell cycle arrest, and an increase in the alkaline phosphatase activity as well, considered a marker of differentiation in colon carcinoma cells. These effects of PB were seen only in FUdR-pretreated cells because PB alone had minimal effect on the expression of these genes. The sequential use of FUdR followed by PB in patients with colon carcinoma should be explored because two subclones of HT29, irrespective of their state of differentiation, respond to this clinically achievable regimen.     

Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells

We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.     

EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.     

Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells

The mitogen-activated protein kinase (MAPK) signal transduction pathway plays an essential role in cell cycle progression and can be activated by many growth factor/mitogen pathways including estrogen. MAPK has also been implicated in ligand-independent activation of estrogen receptor-alpha (ER-alpha). The development of estrogen-independent growth in breast cancer is likely a first step in progression to hormone independence and antiestrogen resistance. We examined MAPK expression and activity in T5-PRF and T5 human breast cancer cells. T5-PRF is an estrogen-nonresponsive cell line developed from T5 cells by chronically depleting the cells of estrogen in long-term culture. MAPK activity measured in vitro was significantly higher (P < 0.05) in T5-PRF compared with T5 cells. Western blot analyses showed increased levels of active dually phosphorylated MAPK in T5-PRF cell extracts compared with T5. The increased activity and expression of MAPK may contribute to the estrogen nonresponsive growth phenotype and ligand-independent activity of ER in T5-PRF cells.     

Growth inhibition of human colon carcinoma cells by combinations of anti-epidermal growth factor-related growth factor antisense oligonucleotides

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self-reactive BI-specific T cell in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1-14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.     

Evidence of cisplatin-induced senescent-like growth arrest in nasopharyngeal carcinoma cells

Cellular senescence is a programmed cell response leading to growth arrest in human diploid fibroblasts. We have shown that a nasopharyngeal carcinoma cell line, CNE1, following treatment by the DNA-damaging agent cisplatin, can undergo cellular senescent-like growth arrest, similar to fibroblasts, judged by cellular morphological changes and the expression of senescence-associated beta-galactosidase (SA-beta-gal). This senescent-like change was dose related; at 0.5 microgram/ml, the percentage of cisplatin-induced SA-beta-gal-positive cells was high (40-96%), and the staining was intense. Higher doses (1.0 and 2.0 micrograms/ml) of cisplatin induced lower SA-beta-gal expression (30-70%), and the process was irreversible. This cisplatin-induced cellular senescent-like response was not due to the inhibition of telomerase activity. Our results indicate that cellular senescent-like pathways exist in nasopharyngeal carcinoma cells and can be induced by cisplatin. Our evidence suggests that cellular senescent-like responses may be a cellular protection mechanism that acts differently in response to different degrees of cellular damage.     

Insulin-like growth factor binding protein production in human follicular thyroid carcinoma cells

OBJECTIVE: The study investigated the value of using national or regional data bases to examine care in a specific hospital. DATA SOURCES: The following data sources were included: (1) the results of the 1992 HCFA analysis of the index hospital for patients hospitalized in fiscal year 1990; (2) the 1989 Medicare Provider Analysis and Review (MEDPAR) file; and (3) clinical information from bypass surgery patients in Wisconsin and from the index hospital. PRINCIPAL FINDINGS: The assessment of the mortality rates in the index hospital for all conditions combined and for CABG patients differed depending on what data base was used and how the data were analysed. The national data were most useful in establishing that the coding practices for all patients and the mortality rate for intra-aortic balloon patients differed between the index hospital and other hospitals. The regional clinical data base for bypass surgery patients was used to establish that the high mortality rates for intra-aortic balloon patients were due to patient selection. CONCLUSIONS: National claims data must be analysed carefully before applying results to an individual hospital. Even a careful analysis is more for raising questions about care at a specific hospital rather than for reaching definitive conclusions.     

Cell proliferation in human prostatic smooth muscle cells involves the modulation of protein kinase C isozymes

We have examined the role of protein kinase C in the regulation of foetal-calf serum-stimulated cell proliferation in human prostatic smooth muscle cells. The data showed that the proliferative effect to foetal-calf serum (10%, v/v) was partially inhibited by 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo (2,3-a) pyrrolo (3,4-c)-carbazole (Go-6976), a selective Ca2+-dependent protein kinase C inhibitor, suggesting that Ca2+-dependent protein kinase C isozymes might play roles in this proliferative regulation. Additionally, foetal-calf serum caused a significant translocation of protein kinase C-betaII and -epsilon from a cytosolic to a membrane distribution. These findings combined with the aforementioned functional experiments suggest that foetal-calf serum-stimulated cell proliferation might involve the activation of protein kinase C-betaII in human prostatic smooth muscle cells; however, the role of protein kinase C-epsilon in mediating cellular functions other than cell proliferation remains further investigation in these cells.     

Cell cycle arrest and release in starfish oocytes and eggs

A neural net-based, semiautomated, interactive computerized cell analysis system (The PAPNET system, Neuromedical Systems, Suffern, NY) was used to examine cells from 138 esophageal smears obtained by lavage, brushings, or balloon from as many patients. From each smear, trained human observers examined 128 cell images selected by the machine. Abnormal cells were identified in all 35 patients with cancer, whether esophageal, gastric, oral, or metastatic. Further, in 11 smears, the displayed images allowed the recognition of effects of radiotherapy and, in 14 smears, the diagnosis of a specific tumor type, such as squamous cell carcinoma (8 patients) or adenocarcinoma (6 patients). In 3 additional cases, the diagnosis of "carcinoma, not further specified," was established. One case of esophageal carcinoma in situ, not previously recognized on a smear or in the biopsy specimen, and one case of gastric adenocarcinoma, not recognized in the smear, were identified in PAPNET-generated images. The possible application of the apparatus to the triage of smears and population screening for esophageal and gastric carcinoma precursors is discussed.     

Constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 induces epithelial dedifferentiation and growth inhibition in madin-darby canine kidney-C7 cells

Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serum-stimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm2 after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G0/G1 to G2/M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serum-stimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.     

Cell cycle regulation of the double stranded RNA activated protein kinase, PKR

The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells. To determine whether endogenous PKR plays a role in cell growth control, we have investigated the regulation of PKR levels and activity during the cell cycle in human glioblastoma T98G cells. The steady-state level of PKR mRNA in T98G cells was highest in growth arrested cells, dropped sharply within 3 h of serum stimulation then gradually increased as cells progressed through G1, reaching a plateau in early S phase. PKR protein level increased following serum stimulation reaching a peak at the G2+M boundary and declining thereafter. In contrast, PKR kinase activity exhibited two peaks, in early G1 and at the G1/S boundary, declining sharply in early S phase. Thus, the activity profile did not follow the protein profile indicating a tight regulation of PKR at the level of activity. In T98G cells expressing the catalytically inactive PKRK296R the dsRNA-induced activation of NF-kappaB and IRF-1 was suppressed and the mutant cells exhibited resistance to stress induced apoptosis. Cell cycle distribution analysis showed that the mutant expressing cells exhibited longer G1 phase and fewer cells engaged in S phase. Furthermore, early passage mouse embryo fibroblasts derived from PKR knockout mice grew more slowly compared with the control cells. Taken together these results suggest that PKR may play a role in cell cycle progression.     

UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase

We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.