Downregulation of Rap1GAP contributes to Ras transformation.

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors.

MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used. For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions. For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (<10%) change in STAT3, Raf-1, and Akt protein levels, while no change was observed for 4E-BP1. The combination treatment resulted in more than 50% decrease of the client protein levels and hypophosphorylation of 4E-BP1 in all MM cell lines. Treatment with 8-Cl-Ado alone resulted in less than 30% decrease in client protein levels as well as a decrease in 4E-BP1 phosphorylation. Cytotoxic evaluation of 8-Cl-Ado in combination with 17-AAG resulted in more than additive cytotoxicity when drugs were combined in a sequential manner. In summary, these data suggest that the mechanism-based combination of agents that target transcription, translation, or decrease cellular bioenergy with 17-AAG results in increase cytotoxicity when compared to the single agents. Such combination strategies may be applied in the clinic since these drugs are established chemotherapeutic agents or currently in clinical trials.

Extended kalman filter for estimation of parameters in nonlinear state-space models of biochemical networks.

It is system dynamics that determines the function of cells, tissues and organisms. To develop mathematical models and estimate their parameters are an essential issue for studying dynamic behaviors of biological systems which include metabolic networks, genetic regulatory networks and signal transduction pathways, under perturbation of external stimuli. In general, biological dynamic systems are partially observed. Therefore, a natural way to model dynamic biological systems is to employ nonlinear state-space equations. Although statistical methods for parameter estimation of linear models in biological dynamic systems have been developed intensively in the recent years, the estimation of both states and parameters of nonlinear dynamic systems remains a challenging task. In this report, we apply extended Kalman Filter (EKF) to the estimation of both states and parameters of nonlinear state-space models. To evaluate the performance of the EKF for parameter estimation, we apply the EKF to a simulation dataset and two real datasets: JAK-STAT signal transduction pathway and Ras/Raf/MEK/ERK signaling transduction pathways datasets. The preliminary results show that EKF can accurately estimate the parameters and predict states in nonlinear state-space equations for modeling dynamic biochemical networks.

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

LMW-E MEDIATES MAMMARY TUMORIGENESIS BY DEREGULATING ACINAR MORPHOGENESIS & GENERATING CANCER STEM CELLS

Cyclin E is the regulatory subunit of the cyclin E/CDK2 complex that mediates the G1-S phase transition. N-terminal cleavage of cyclin E by elastase in breast cancer generates two low molecular weight (LMW) isoforms that exhibit both enhanced kinase activity and resistance to p21 and p27 inhibition compared to fulllength cyclin E. Clinically, approximately 27% of breast cancer patients overexpress LMW-E and associate with poor survival. Therefore, we hypothesize that LMW-E disrupts normal mammary acinar morphogenesis and serves as the initial route into breast tumor development. We first demonstrate that LMW-E overexpression in non-tumorigenic hMECs is sufficient to induce tumor formation in athymic mice significantly more than overexpression of full-length cyclin E and requires CDK2- associated kinase activity. Further in vivo passaging of these tumors augments LMW-E expression and tumorigenic potential. When subjected to acinar morphogenesis in vitro, LMW-E mediates significant morphological disruption by generating hyperproliferative and multi-acinar complexes. Proteomic analysis of patient tissues and tumor cells with high LMW-E expression reveals that the activation of the b-Raf-ERK1/2-mTOR pathway in concert with high LMW-E expression predicts poor patient survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (b-raf inhibitor) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing the G1/S cell cycle arrest. In addition, the LMW-E-expressing tumor cells exhibit phenotypes characteristic of the EMT and enhanced cellular invasiveness. These tumor cells also enrich for cells with CSC phenotypes such as increased CD44hi/CD24lo population, enhanced mammosphere formation, and upregulation of ALDH expression and enzymatic activity. Furthermore, the CD44hi/CD24lo population also shows positive correlation with LMW-E expression in both the tumor cell line model and breast cancer patient samples (p<0.0001 & p=0.0435, respectively). Combination treatment using doxorubicin and salinomycin demonstrates synergistic cytotoxic effects in cells with LMW-E expression but not in those with full-length cyclin E expression. Finally, ProtoArray microarray identifies Hbo1 as a novel substrate of the cyclin E/CDK2 complex and its overexpression results in enrichment for CSCs. Collectively, these data emphasize the strong oncogenic potential of LMW-E in mammary tumorigenesis and suggest possible therapeutic strategies to treat breast cancer patients with high LMW-E expression.

Phosphorylation in the regulation of muscle-specific transcription

The contents of this dissertation include studies on the mechanisms by which FGF and growth factor down-stream kinases inactivate myogenin; characterization of myogenin phosphorylation and its role in regulation of myogenin activity; analysis the C-terminal transcriptional activation domain of myogenin; studies on the nuclear localization of myogenin and characterization of proteins that interact with PKC.^ Activation of muscle transcription by the MyoD family requires their heterodimerization with ubiquitous bHLH proteins such as the E2A gene products E12 and E47. I have shown that dimerization with E2A products potentiates phosphorylation of myogenin at serine 43 in its amino-terminus and serine 170 in the carboxyl-terminal transcription activation domains. Mutations of these sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. Consistent with the role of phosphorylation at serine 170, analysis of the carboxyl-terminal transcriptional activation domain by deletion has revealed a stretch of residues from 157 to 170 which functions as a negative element for myogenin activity.^ In addition to inducing phosphorylation of myogenin, E12 also localizes myogenin to the nucleus. The DNA binding and dimerization mutants of myogenin show various deficiencies in nuclear localization. Cotransfection of E12 with the DNA binding mutants, but not a dimerization mutant, greatly enhances their nuclear binding. These data suggest that the nuclear localization signal is located in the DNA binding region and myogenin can also be nuclear localized by virtue of dimerizing with a nuclear protein.^ FGF is one of the most potent inhibitors of myogenesis and activates many down-stream pathways to exert its functions. One of these pathway is the MAP kinase pathway. Studies have shown that Raf-1 and Erk-1 kinase inactivate transactivation by myogenin and E proteins independent of DNA binding. The other is the PKC pathway. In transfected cells, FGF induces phosphorylation of thr-87 that maps to the previously identified PKC sites in the DNA binding domain of myogenin. Myogenin mutant T-N87 could resist the inhibition directed to the bHLH domain by FGF, suggesting that FGF inactivates myogenin by inducing phosphorylation of this site. In C2 myotubes, where FGF receptors are lost, the phosphatase inhibitor, okadaic acid, and phorbal ester PdBu, can also induce the phosphorylation of thr-87. This result supports the previous observation and suggests that in myotubes, other mechanisms, such as innervation, may inactivate myogenin through PKC induced phosphorylation.^ Many functions of PKC have been well documented, yet, little is known about the activators or effectors of PKC or proteins that mediate PKC nuclear localizations. Identification of PKC binding proteins will help to understand the molecular mechanism of PKC function. Two proteins that interact with the C kinase (PICKS) have been characterized, PICK-1 and PICK-2. PICK1 interacts with two conserved regions in the catalytic domain of PKC. It is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK2 is a novel protein with homology to the heat shock protein family. It interacts extensively with the catalytic domain of PKC and is localized in the cytoplasm in a punctate pattern. PICK1 and PICK2 may play important roles in mediating the actions of PKC. ^

The role of the sonic hedgehog signaling pathway in epidermal homeostasis

Non-melanoma skin cancer is the most frequently diagnosed malignancy in the United States of which basal cell carcinoma (BCC) accounts for 65%. It has recently been determined that deregulation of the sonic hedgehog (shh) pathway leads to the development of BCC. Shh, gli-1, gli-2 gli-3, ptc and smo are overexpressed in BCC and overexpression of these genes in the epidermis results in formation of BCC-like tumors. Despite these observations, the mechanisms by which the pathway controls epidermal homeostasis and the development of the malignant phentotype are unknown. This study assessed the role of the shh pathway in epidermal homeostasis through regulation of apoptosis and differentiation. ^ The anti-apoptotic protein, bcl-2 is overexpressed in BCC, however transcriptional regulators of bcl-2 in the epidermis are unknown. Transient transfection of primary keratinocytes with gli-1 resulted in an increase of bcl-2 expression. Database analysis revealed seven candidate gli binding sites on the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. An N-terminal mutant of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. The region −428 to −420 was found to be important for gli-1 regulation through gel shift, luciferase assays and site-directed mutagenesis. ^ In order to assess the ability of the shh pathway to regulate keratinocyte differentiation, HaCaT keratinocytes overexpressing sonic hedgehog, were grown in organotypic raft culture. Overexpression of shh induced a basal cell phenotype compared to vector control, as evidenced by transmural staining of cytokeratin 14 and altered Ki67 staining. Shh also induced keratinocyte invasion into the underlying collagen. This was associated with increased phosphorylation of EGFR, jnk and raf and increased expression of c-jun, mmp-9 and Ki67. Interestingly, shh overexpression in HaCaTs did not induce the typical downstream effects of shh signaling, suggesting a gli-independent mechanism. Sonic hedgehog's ability to induce an invasive phenotype was found to be dependent on activation of the EGF pathway as inhibition of EGFR activity with AG1478 and c-225 was able to reduce the invasiveness of HaCaT shh keratinocytes, whereas treatment with EGF augmented the invasiveness of the HaCaT shh clones. ^ These studies reveal the importance of the sonic hedgehog pathway in epidermal homeostasis by regulation of apoptosis through bcl-2, and control of keratinocyte differentiation and invasion through activation of the EGF pathway. They further suggest potential mechanisms by which deregulation of the shh pathway may lead to the development of the malignant phenotype. ^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

The role of STAT3 in the emergency neutrophil response

Neutrophils are an essential component of innate immunity, serving to provide an immediate response to microbial invasion. In response to emergency situations such as an infection, serum levels of granulocyte colony-stimulating factor (G-CSF) are induced, causing a boost in neutrophil production and a rapid mobilization of bone marrow neutrophils to the blood, where they can circulate to clear foreign pathogens. Signal transducer and activator of transcription 3 (STAT3) is a principal downstream signaling intermediate of the G-CSF receptor. Mice null for STAT3 are embryonic lethal; therefore, to examine the role that STAT3 has in granulocytic development and function in vivo, we utilized a conditional knockout mouse that deletes functional STAT3 in the hematopoietic system (referred to herein as STAT3-deficient). Using this model, we show that STAT3 is required for G-CSF-induced expansion of granulocytic progenitor cells within the bone marrow and for acute G-CSF-dependent neutrophil mobilization into the blood. Thus, STAT3 has a critical role in the immediate G-CSF-response in vivo. Sustained G-CSF exposure causes skewed granulocytic production and mobilization in STAT3-deficient mice, suggesting an atypical granulocytic developmental pathway. To determine if STAT3-deficient neutrophils were functional, we examined neutrophil chemotaxis, since neutrophil function relies on proper chemoattractant-induced migration to infected tissue sites. STAT3-deficient neutrophils have impaired chemotaxis in response to the potent neutrophil chemoattractants MIP-2 and KC, both ligands for the chemokine receptor CXCR2. Additionally, STAT3-deficient mice have a defect in NIIP-2-induced acute neutrophil mobilization in vivo. Chemotaxis in response to fMLP and SDF-1, which utilize distinct seven-transmembrane chemokine receptors, was similar between wild type and STAT3-deficient neutrophils, suggesting that STAT3 specifically regulates CXCR2-mediated migration. MIP-2-induced activation of the Raf/MEK/ERK signaling cascade, which we show is required for MIP-2-dependent neutrophil chemotaxis, was impaired in STAT3-deficient neutrophils. Interestingly, acute G-CSF administration induced CXCR2 expression and Raf/MEK/ERK activation in neutrophils from wild type mice, whereas these responses were abrogated in neutrophils from STAT3-deficient mice. Thus, STAT3 regulation of CXCR2 functions may also contribute to STAT3's control of the acute G-CSF mobilization response. These combined results place STAT3 as a critical intermediate in neutrophil migration and G-CSF-induced neutrophil production responses required for emergency granulopoiesis. ^

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform.

Transcriptional regulation of the rat {\it mdr1b\/} gene

The expression of P-glycoproteins encoded by the mdr gene family is associated with the emergence of multidrug-resistance phenotype in animal cells. This gene family includes two members, MDR1 and MDR2, in humans, and three members, mdr1a, mdr1b, and mdr2, in rodents. Among them, the rat mdr1b is known to be highly activated during hepatocarcinogenesis, and its expression is sensitive to the treatment with growth factors, cytotoxic drugs, as well as other physical or chemical stresses. It is believed that the transcriptional regulation plays an important role in above events, however little has been known about mechanisms involved.^ To elucidate how mdr1b expression is regulated, we isolated the genomic sequence of the rat mdr1b and functionally dissected its 5$\prime$ promoter region. Our results demonstrated that: (1) the transcription start site of the rat mdr1b is identical to that of the murine mdr1b homologue; (2) a palindromic sequence from bp $-$189 to $-$180 bp is essential for the basal promoter function of the rat mdr1b, and binds to a specific protein that appears to be a novel transcription factor implicated in the regulation of the rat mdr1b expression; (3) a NF-$\kappa$B-binding site from bp $-$167 to $-$159 is also involved in the basal promoter function. The p65/p50 subunits of the NF-$\kappa$B and raf-1 kinase are implicated in the insulin-inducible promoter activity of the mdr1b, suggesting the important role of NF-$\kappa$B in the regulation of the mdr1b by growth factors; (4) a p53-binding site from bp $-$199 to $-$180 is not only essential for the basal promoter activity but also responsible for the induction of mdr1b by cytotoxic agents. In addition, we provided evidence showing that endogenous mdr1b expression can be modulated by wild-type p53. On the basis of these findings, a model of transcriptional regulation of the rat mdr1b was proposed. ^

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^