Maintain Cell Cycle Research Articles

Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis

Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G(1) cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21(Cip). Notably, concomitant genetic inactivation of p53 or p21(Cip) indicates that Hdac1 and Hdac2 regulate p53-p21(Cip)-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors.

Transcriptional Repression of miR-34 Family Contributes to p63-Mediated Cell Cycle Progression in Epidermal Cells

p63, a p53 family member, is highly expressed in the basal proliferative compartment of the epidermis and its expression has been correlated with the growth ability and regenerative capacity of keratinocytes. In this study we report a mechanism through which p63 maintains cell cycle progression by directly repressing miR-34a and miR-34c. In the absence of p63, increased levels of miR-34a and miR-34c were observed in primary keratinocytes and in embryonic skin, with concomitant G1-phase arrest and inhibition of the cell cycle regulators cyclin D1 and cyclin-dependent kinase 4 (Cdk4). p63 directly bound to p53-consensus sites in both miR-34a and miR-34c regulatory regions and inhibited their activity. Concomitant downregulation of miR-34a and miR-34c substantially restored cell cycle progression and expression of cyclin D1 and Cdk4. Our data indicate that specific miR-34 family members have a significant role downstream of p63 in controlling epidermal cell proliferation.

Abstract 3870: Distinct and cooperative functions of the RB and p53 tumor suppressors in genotoxic response and tumorigenesis

Abstract The compound inactivation of RB and p53 is a frequent occurrence in human cancers. At a molecular level, the RB and p53 pathways intersect at multiple points that have critical implications for cell cycle control and tumorigenesis. To examine the cooperation of these proteins in the response to genotoxic damage, we used a combination of genetic deletion and dominant negative strategies in vitro, and liver-specific genetic deletion in vivo. In vitro, RB proficiency was sufficient to maintain cell cycle checkpoint in response to genotoxic stress, independent of p53 status. In contrast, RB-deficient cells displayed deregulated cell growth, resulting in aggressive recovery from DNA damage. Strikingly, while disruption of the RB and p53 pathways alone does not result in transformation, the RB-deficient, recurred cell populations were tumorigenic. These in vitro findings indicate that RB is a critical suppressor of tumorigenesis, beyond that of p53, and a significant factor in the evolution of cells during genotoxic challenge. To further examine the cooperation of RB and p53 in vivo, a model of liver tumorigenesis was employed. Interestingly, these in vivo studies confirmed that while compound inactivation of the RB and p53 pathways in liver tissue promoted deregulated cell cycle control, this was not sufficient for transformation. However, loss of RB and p53 cooperated in promoting aberrant cell proliferation and mitosis in the presence of genotoxic challenge by DEN (diethyl-nitrosamine), ultimately resulting in enhanced tumorigenesis. Strikingly, loss of RB alone promoted S-phase entry as a result of genotoxic challenge, but there was an uncoupling of this event from productive mitosis. In contrast, the combined loss of RB/p53 resulted in further deregulation of DNA replication that was accompanied by a productive mitotic entry. These data indicate that, while both RB and p53 can contribute to appropriate replicative control, p53 plays a dominant role in mediating G2/M regulation. Furthermore, RB/p53-deficient livers displayed rapid progression to highly aggressive HCC within the span of six months, which was not observed in either RB-deficient or p53-deficient livers. Combined, these studies indicate that RB and p53 play distinct and cooperating roles in modulating response to genotoxic stress and tumorigenesis. Discovering the mechanism behind this cooperation could uncover a critical facet of tumor etiology and provide a means by which to treat this complex disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3870.

Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

Female mice lacking the transcription factor C/EBPbeta are infertile and display markedly reduced estrogen (E)-induced proliferation of the uterine epithelial lining during the reproductive cycle. The present study showed that E-stimulated luminal epithelial cells of a C/EBPbeta-null uterus are able to proceed through the G1 phase of the cell cycle before getting arrested in the S phase. This cell cycle arrest was accompanied by markedly reduced levels of expression of E2F3, an E2F family member, and a lack of nuclear localization of cyclin E, a critical regulator of cdk2. An increased nuclear accumulation of p27, an inhibitor of the cyclin E-cdk2 complex, was also observed for the mutant epithelium. Gene expression profiling of C/EBPbeta-null uterine epithelial cells revealed that the blockade of E-induced DNA replication triggers the activation of several well-known components of the DNA damage response pathway, such as ATM, ATR, histone H2AX, checkpoint kinase 1, and tumor suppressor p53. The activation of p53 by ATM/ATR kinase led to increased levels of expression of p21, an inhibitor of G1-S-phase progression, which helps maintain cell cycle arrest. Additionally, p53-dependent mechanisms contributed to an increased apoptosis of replication-defective cells in the C/EBPbeta-null epithelium. C/EBPbeta, therefore, is an essential mediator of E-induced growth and survival of uterine epithelial cells of cycling mice.

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G 2/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.

Cell Cycle Control of Kaposi's Sarcoma-Associated Herpesvirus Latency Transcription by CTCF-Cohesin Interactions

Kaposi's sarcoma-associated herpesvirus (KSHV) latency is characterized by the highly regulated transcription of a few viral genes essential for genome maintenance and host cell survival. A major latency control region has been identified upstream of the divergent promoters for the multicistronic transcripts encoding LANA (ORF73), vCyclin (ORF72), and vFLIP (ORF71) and for the complementary strand transcript encoding K14 and vGPCR (ORF74). Previous studies have shown that this major latency control region is occupied by the cellular chromatin boundary factor CTCF and chromosome structural maintenance proteins SMC1, SMC3, and RAD21, which comprise the cohesin complex. Deletion of the CTCF-cohesin binding site caused an inhibition of cell growth and viral genome instability. We now show that the KSHV genes regulated by CTCF-cohesin are under cell cycle control and that mutation of the CTCF binding sites abolished cell cycle-regulated transcription. Cohesin subunits assembled at the CTCF binding sites and bound CTCF proteins in a cell cycle-dependent manner. Subcellular distribution of CTCF and colocalization with cohesins also varied across the cell cycle. Ectopic expression of Rad21 repressed CTCF-regulated transcription of KSHV lytic genes, and a Rad21-CTCF chimeric protein converted CTCF into an efficient transcriptional repressor of KSHV genes normally activated in the G(2) phase. We conclude that cohesins interact with CTCF in mid-S phase and repress CTCF-regulated genes in a cell cycle-dependent manner. We propose that the CTCF-cohesin complex plays a critical role in regulating the cell cycle control of viral gene expression during latency and that failure to maintain cell cycle control of latent transcripts inhibits host cell proliferation and survival.

Directed expression of dominant-negative p73 enables proliferation of cardiomyocytes in mice

Previous studies have shown that p53 plays an important role in maintaining cell cycle arrest of cardiomyocytes, which might account for the inability of human hearts to regenerate adequately after injury. Therefore, inhibition of p53 represents an attractive strategy to restore cell cycle progression in cardiomyocytes although such an approach is hampered by the potential danger of concomitant tumor induction. During normal development, N-terminal truncated isoforms of the p53-related protein p73 are naturally occurring antagonists of p53 and p73, which are not related to tumor induction. We have generated recombinant adenoviruses encoding dominant-interfering p73 (Ad-p73DD) to inhibit p53/p73 in murine hearts at different developmental stages. We found that the expression of p73DD(wt) in newborn mice led to the increase of the relative heart weights after 14 days which is paralleled by a significant increase of proliferating cardiomyocytes as seen by ICC (BrdU-incorporation, phosphorylation of histone3, expression of AuroraB) without induction of apoptosis. Stimulation of cell cycle progression in cardiomyocytes went along with a significant down-regulation of the p53-dependent cdk-inhibitor p21WAF both on mRNA and protein level. Furthermore, mRNA levels and protein expression of D-type cyclins and cyclins A, B2, and E were selectively increased after expression of p73DD. We further show that the cell cycle entry of cardiomyocytes is not restricted to neonatal hearts but is also found in adult mouse hearts 5 days after intramyocardial injection of Ad-p73DD. Taken together we reason that directed expression of dominant-negative p73 might be utilized to stimulate proliferation of cardiomyocytes to improve cardiac regeneration.

IL-21 promotes survival and maintains a naive phenotype in human CD4+ T lymphocytes

IL-21 is a key T-cell growth factor (TCGF) involved in innate and adaptive immune response. It contributes to the proliferation of naive, but not memory T lymphocytes. However, the full spectrum of IL-21 activity on T cells remains unclear. Here, we demonstrate that IL-21 primarily maintains the expression of specific naive cell surface markers such as CD45RA, CD27, CD62L and CCR7 on human CD4(+) T lymphocytes and that the expression of CCR7 induces cell migration by means of CCL21 chemoattraction. These effects contrast with those of IL-2 which induced the marked proliferation of CD4(+) T lymphocytes, leading to an activated-memory phenotype. Nevertheless, IL-21 maintained cell cycle activation and expression of proliferation markers, including proliferating cell nuclear antigen and Ki-67, and triggered T-cell proliferation via TCR and co-stimulation pathways. Unlike IL-2, IL-21 decreased the expression of the anti-apoptotic Bcl-2 protein, which correlated with the absence of activation of the phosphatidylinositol 3'-kinase/Akt signaling pathway. Thus, IL-21 is a TCGF whose function is the preservation of a pool of CD4(+) T lymphocytes in a naive phenotype, with a low proliferation rate but with the persistence of cell cycling proteins and cell surface expression of CCR7. These findings strongly suggest that IL-21 plays a part in innate and adaptive immune response owing to homeostasis of T cells and their homing to secondary lymphoid organs.

Estrogens decrease γ-ray–induced senescence and maintain cell cycle progression in breast cancer cells independently of p53

Sequential administration of radiotherapy and endocrine therapy is considered to be a standard adjuvant treatment of breast cancer. Recent clinical reports suggest that radiotherapy could be more efficient in association with endocrine therapy. The aim of this study was to evaluate the estrogen effects on irradiated breast cancer cells (IR-cells). Using functional genomic analysis, we examined the effects of 17-beta-estradiol (E(2), a natural estrogen) on MCF-7 breast cancer cells. Our results showed that E(2) sustained the growth of IR-cells. Specifically, estrogens prevented cell cycle blockade induced by gamma-rays, and no modification of apoptotic rate was detected. In IR-cells we observed the induction of genes involved in premature senescence and cell cycle progression and investigated the effects of E(2) on the p53/p21(waf1/cip1)/Rb pathways. We found that E(2) did not affect p53 activation but it decreased cyclin E binding to p21(waf1/cip1) and sustained downstream Rb hyperphosphorylation by functional inactivation of p21(waf1/cip1). We suggest that Rb inactivation could decrease senescence and allow cell cycle progression in IR-cells. These results may help to elucidate the molecular mechanism underlying the maintenance of breast cancer cell growth by E(2) after irradiation-induced damage. They also offer clinicians a rational basis for the sequential administration of ionizing radiation and endocrine therapies.

Effect of salt hyperosmotic stress on yeast cell viability

During fermentation for ethanol production, yeasts are subjected to different kinds of physico-chemical stresses such as: initially high sugar concentration and low temperature; and later, increased ethanol concentrations. Such conditions trigger a series of biological responses in an effort to maintain cell cycle progress and yeast cell viability. Regarding osmostress, many studies have been focused on transcriptional activation and gene expression in laboratory strains of Saccharomyces cerevisiae. The overall aim of this present work was to further our understanding of wine yeast performance during fermentations under osmotic stress conditions. Specifically, the research work focused on the evaluation of NaCl-induced stress responses of an industrial wine yeast strain S. cerevisiae (VIN 13), particularly with regard to yeast cell growth and viability. The hypothesis was that osmostress conditions energized specific genes to enable yeast cells to survive under stressful conditions. Experiments were designed by pretreating cells with different sodium chloride concentrations (NaCl: 4%, 6% and 10% w/v) growing in defined media containing D-glucose and evaluating the impact of this on yeast growth and viability. Subsequent fermentation cycles took place with increasing concentrations of D-glucose (20%, 30%, 40% w/v) using salt-adapted cells as inocula. We present evidence that osmostress induced by mild salt pre-treatments resulted in beneficial influences on both cell viability and fermentation performance of an industrial wine yeast strain.

Tissue-Specific Deletion of the Retinoblastoma Protein in the Pancreatic β-Cell Has Limited Effects on β-Cell Replication, Mass, and Function

Animal studies show that G(1/S) regulatory molecules (D-cyclins, cdk-4, p18, p21, p27) are critical for normal regulation of beta-cell proliferation, mass, and function. The retinoblastoma protein, pRb, is positioned at the very end of a cascade of these regulatory proteins and is considered the final checkpoint molecule that maintains beta-cell cycle arrest. Logically, removal of pRb from the beta-cell should result in unrestrained beta-cell replication, increased beta-cell mass, and insulin-mediated hypoglycemia. Because global loss of both pRb alleles is embryonic lethal, this hypothesis has not been tested in beta-cells. We developed two types of conditional knockout (CKO) mice in which both alleles of the pRb gene were inactivated specifically in beta-cells. Surprisingly, although the pRb gene was efficiently recombined in beta-cells of both CKO models, changes in beta-cell mass, beta-cell replication rates, insulin concentrations, and blood glucose levels were limited or absent. Other pRb family members, p107 and p130, were not substantially upregulated. In contrast to dogma, the pRb protein is not essential to maintain cell cycle arrest in the pancreatic beta-cell. This may reflect fundamental inaccuracies in models of beta-cell cycle control or complementation for pRb by undefined proteins.

Dominant Role of Raf/MEK/ERK Signaling in Hematopoietic Cell Cycle Progression, Prevention of Apoptosis and Drug Resistance.

The Ras/Raf/MEK/ERK and PI3K/PTEN/AKT signaling cascades and p53 play critical roles in the transmission of signals from growth factor receptors to regulate gene expression, growth, malignant transformation and drug resistance. We have investigated the roles these two pathways play in cell cycle progression, prevention of apoptosis and drug resistance in cytokine-dependent FL5.12 hematopoietic cells and derivative FL/Akt:ER+Raf-1:AR cells conditionally transformed to grow in response to Akt and Raf activation. The effects of these two pathways on cell cycle progression and sensitivity to chemotherapeutic drugs could be more definitively examined in FL/Akt:ER+Raf-1:AR cells where it is possible to induce either Akt or Raf by themselves or together by the addition of tamoxifen or testosterone. Raf-1 was approximately 8-fold more effective than Akt in maintaining cell cycle progression and preventing apoptosis induced by the chemotherapeutic drugs. However, growth in the presence of chemotherapeutic drugs was enhanced significantly when both Akt and Raf were activated, documenting roles for both Raf and Akt in drug resistance. Drug resistant cells were isolated from both parental and derivative lines in the presence of 10 to 100 nM doxorubicin. Doxorubicin resistant cells were also cross resistant to paclitaxel and daunomycin but not to cisplatin and 5-fluoruracil which are transported by different drug pumps, however, drug resistance did not appear to be mediated by MDR-1/MRP-1 as neither elevated drug pump activity nor higher levels of proteins or mRNAs encoding these proteins were detected, while they were detected in appropriate controls. Drug resistant FL5.12 cells (which lacked any introduced gene) displayed elevated expression of activated ERK, hypersensitivity to Raf and MEK inhibitors and decreased caspase 3 cleavage following doxorubicin treatment. The drug resistance of FL5.12 cells could be further increased 5-fold by introduction of an activated MEK1 gene into the cells and likewise suppressed 8-fold by introduction of a dominant negative (DN) MEK1 gene. Introduction of activated MEK1 also resulted in a further 10-fold decrease in sensitivity to the induction of apoptosis stimulated by doxorubicin treatment as measured by Annexin V/PI binding and Caspase 3 cleavage. These results provide evidence for roles of MEK/ERK in hematopoietic drug resistance. p53 was also involved in drug sensitivity as introduction of DN p53 into FL5.12 cells, which normally express wild-type p53, increased their doxorubicin resistance approximately 3-fold and importantly sensitivity to MEK inhibitors was also determined to be p53-dependent. In summary, our results indicate that the Raf/MEK/ERK pathway is critical in chemotherapy-induced drug resistance and provide rationale for combinations of chemotherapy and Raf/MEK/ERK inhibitors.

Stromal Cell Line Enhances Survival of Myeloid Leukemia Cells by Regulating the Expression of bcl-2 Family Genes and Caspase-3.

Approximately 20–30% acute myeloid leukemia (AML) patients are not cured by traditional chemotherapy. The possible explanation is that residual leukemia cells are harbored in association with stromal cells in the bone marrow, which causes late relapse of leukemia. This study was designed to investigate interactions between marrow stromal microenvironment and leukemia cells. AML cells were cultured in different conditions with or without serum, Ara-c, the monolayers or the conditioned medium of a stromal cell line HESS-5. Apoptosis frequencies, cell viability, cell cycle distribution, DNA fragment, and bcl-2/bcl-xl gene as well as caspase-3 expression were determined by Typan blue exclusion, MTT assay, flow cytometry analysis, PCR and Western blot respectively. Direct contact of HL-60 cells with HESS-5 significantly inhibited serum-deprivation- or Ara-c-induced apoptosis of HL-60 cells, and resulted in a significant increase of short-term proliferation and viability. Soluble factors had negligible effect. The interaction of HL-60 with fibronectin did not confer a significant resistance to serum-deprivation and Ara-c agent. HESS-5 cells were also able to maintain cell cycle progression of HL-60 during chemotherapy exposure. HESS-5 cells-mediated apoptosis inhibition was associated with increased bcl-2/bcl-xl expression and down-regulation of caspase-3 in primary myeloid leukemia cells. These results indicate that stromal cells enhance the survival of myeloid leukemia cells by regulating bcl-2 family genes as well as caspase-3 protein and suggest that specific interactions between stromal cells and leukemia cells may enhance the resistance of leukemia cells to chemotherapy.

Selective Chk1 inhibitors differentially sensitize p53‐deficient cancer cells to cancer therapeutics

The majority of cancer therapeutics induces DNA damage to kill cells. Normal proliferating cells undergo cell cycle arrest in response to DNA damage, thus allowing DNA repair to protect the genome. DNA damage induced cell cycle arrest depends on an evolutionarily conserved signal transduction network in which the Chk1 kinase plays a critical role. In mammalian cells, the p53 and RB pathways further augment the cell cycle arrest response to prevent catastrophic cell death. Given the fact that most tumor cells suffer defects in the p53 and RB pathways, it is likely that tumor cells would depend more on the Chk1 kinase to maintain cell cycle arrest than would normal cells. Therefore Chk1 inhibition could be used to specifically sensitize tumor cells to DNA-damaging agents. We have previously shown that siRNA-mediated Chk1 knockdown abrogates DNA damage-induced checkpoints and potentiates the cytotoxicity of several DNA-damaging agents in p53-deficient cell lines. In this study, we have developed 2 potent and selective Chk1 inhibitors, A-690002 and A-641397, and shown that these compounds abrogate cell cycle checkpoints and potentiate the cytotoxicity of topoisomerase inhibitors and gamma-radiation in p53-deficient but not in p53-proficient cells of different tissue origins. These results indicate that it is feasible to achieve a therapeutic window with 1 or more Chk1 inhibitors in potentiation of cancer therapy based on the status of the p53 pathway in a wide spectrum of tumor types.

Transcriptional blackjack with p21: Figure 1.

Transcriptional regulation by RNA polymerase II (RNA Pol II) is an amalgamated process requiring the collective action of a large number of transcription factors to ensure proper synthesis of messenger RNA (mRNA). Several RNA Pol II elongation factors and proper phosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of Pol II have been shown to be essential for transcription when RNA Pol II enters its processive elongation stage. In a study reported in the previous issue of Genes & Development, Emerson and colleagues (Gomes et al. 2006) demonstrated an exception to this rule. Transcription of p21, a target gene within the p53 pathway, bypasses the requirements for RNA Pol II elongation factors (including P-TEFb) and CTD phosphorylation for its p53-dependent transcription. Their data indicate the presence of a cellular compensatory mechanism for several of the p53 target genes to ensure a transcriptional response under heightened cellular stress. Mammalian cells are bombarded with a host of cellular stresses and require specialized mechanisms to maintain their integrity. The p53 protein is a sequence-specific DNA-binding transcription factor that regulates expression of numerous genes involved in cell cycle arrest and apoptosis, and is regarded as the cell’s cornerstone of defense against stresses such as DNA damage, oncogene activation, and oxidative stress (Takimoto and El-Deiry 2001; Taylor and Stark 2001). Several genes involved in the DNA damage response pathway contain p53-binding elements upstream of their promoters. In response to chemical or irradiation-induced DNA damage, binding of p53 to target genes occurs rapidly to shut off cell cycle progression and allow for recovery. There are a variety of p53-dependent pathways that can maintain cell cycle blocks (Bunz et al. 1998; Agami and Bernards 2000; Laronga et al. 2000; Vousden 2000; Gartel and Tyner 2002). Induction of cell cycle arrest by p53 is regulated by the level of transcriptional activation of the p21/WAF1/CIP1 gene (Espinosa and Emerson 2001; Takimoto and El-Deiry 2001; Espinosa et al. 2003). Once p21 (a potent inhibitor of cyclindependent kinases) is expressed, the cells become arrested (Agami and Bernards 2000). The topoisomerase II inhibitor doxorubicin induces double-stranded DNA breaks and is also a potent stimulator of p53 activity (Ravizza et al. 2004). The results presented by Gomes et al. (2006) in the previous issue of Genes & Development give new insight into the complicated mechanisms cells use to respond to various stresses, and strongly suggest the existence of highly specialized cellular responses that are specific for each type of stress-inducing factor.

Beyond the cell cycle: A new role for Cdk6 in differentiation

Over 10 years ago, cdk6 was identified as a new member in a family of vertebrate cdc-2 related kinases. This novel kinase was found to partner with the D-type cyclins and to possess pRb kinase activity in vitro and has since been understood to function solely as a pRb kinase in the regulation of the G(1) phase of the cell cycle. In the past 2 years, several independent studies in multiple cell types have indicated a novel role for cdk6 in differentiation. For example, cdk6 expression must be reduced to allow proper osteoblast and osteoclast differentiation, forced cdk6 expression blocked differentiation of mouse erythroid leukemia cells and cdk6 expression in primary astrocytes favors the expression of progenitor cell markers. Since exit from the cell cycle is a necessary step in terminal differentiation, down-regulation of a mitogenic factor may be expected in this process, however it is surprising that this association has not been previously uncovered and that it is apparently not shared with cdk4, long understood to be a functional homolog of cdk6. The mechanism of cdk6 function in differentiation is not understood, but it may extend beyond the established role of cdk6 as a pRb kinase. As this story unfolds it will be important to discover if the function of cdk6 in differentiation is pRb-dependent or pRb-independent, since pRb has long been established as a key factor in initiating and maintaining cell cycle exit during differentiation.

Lactoferrin interaction with retinoid signaling: Cell growth and apoptosis in mammary cells

Lactoferrin (Lf) is a multifunctional iron-binding protein that was first identified in mammary secretions, but is synthesized by most mammalian tissues. The protein has a signal sequence that dictates secretion; it also has a nuclear localization sequence that facilitates entry into the cell nucleus. The mechanism of the latter action is currently unknown, but is thought to occur via a Lf receptor. Lactoferrin content of mammary tissue and secretions varies with developmental state; it is synthesized in mammary tissue at high levels during both pregnancy and involution, and during mammary infections. Using fluorescent (FITC)-labeled holo-bLf, we show that bovine primary epithelial cells and MCF-7 breast cancer cells do not translocate the exogenously added Lf to the nucleus after culture in serum free media (SFM). However, the supplementation of SFM with 1 μM all-trans retinoic acid (atRA) caused breast cancer cells to gain the capacity to take up labeled bLf into the cell nucleus. Primary bovine mammary cells (MeBo) exhibited similar capacity in culture. This suggests that in addition to Lf, one or more components modulated by atRA, are necessary for nuclear translocation to occur. Transfection experiments with atRA treated MCF-7 cells containing retinoic acid response element reporter constructs showed that the extracellular application of lactoferrin alters reporter gene expression. Lactoferrin increased a DR5 luciferase response element in a dose-dependent manner only when atRA was applied. Immunocytochemical markers for the cell cycle (Ki67) and apoptotic events (Caspase-3 and PARP-85) showed that lactoferrin alters the atRA-induced phenotype, blocking apoptosis and maintaining cell cycle activity in both MCF-7 and MeBo cells in the presence of 1 μM atRA. We propose that nuclear lactoferrin interacts with retinoic acid signaling pathways in cells and alters/blocks the signals so that cells remain in the cell cycle and/or do not enter the apoptotic pathway.

Regulation of the G2/M Transition in Xenopus Oocytes by the cAMP-dependent Protein Kinase

Vertebrate oocytes are arrested in G(2) phase of the cell cycle at the prophase border of meiosis I. Progesterone treatment of Xenopus oocytes releases the G(2) block and promotes entry into the M phases of meiosis I and II. Substantial evidence indicates that the release of the G(2) arrest requires a decrease in cAMP and reduced activity of the cAMP-dependent protein kinase (PKAc). It has been reported and we confirm here that microinjection of either wild type or kinase-dead K72R PKAc inhibits progesterone-dependent release of the G(2) arrest with equal potency and that inhibition can be reversed by a second injection of the heat-stable inhibitor of PKAc, PKI. However, a mutant enzyme predicted to be completely kinase-dead from the crystal structure of PKAc, K72H PKAc, was much less inhibitory when carrying additional mutations that block interaction with either type I or type II regulatory subunit. Moreover, inhibition by K72H PKAc was reversed by PKI at a 30-fold lower concentration and with more rapid kinetics compared with wild type PKAc. K72R PKAc was found to have low but detectable activity after incubation in an oocyte extract. These results indicate that inhibition of the progesterone-dependent G(2)/M transition in oocytes after microinjection of dead PKAc reflects either low residual activity or binding to regulatory subunits with a resulting net increase in the level of endogenous wild type PKAc. Consistent with this hypothesis, the induction of mitosis in Xenopus egg extracts by the addition of cyclin B was blocked by wild type PKAc but not by K72H PKAc. The identification of substrates for PKAc that maintain cell cycle arrest in G(2) remains an important goal for future work.

Interferon‐γ inhibits cell cycle exit in differentiating oligodendrocyte progenitor cells

The developmental processes of the oligodendrocyte progenitor cell (OPC) lineage that are targeted by interferon-gamma (IFN-gamma) were studied in primary rat OPC cultures. Under conditions of thyroid hormone-mediated oligodendrocyte differentiation, IFN-gamma produced a dose-dependent apoptotic response in OPCs. The lowest dose tested (15 ng/ml or 75 U/ml) was nonapoptotic, but activated detectable STAT1 DNA-binding. At this dose, IFN-gamma reduced the percentage of mature O1+ cells and increased the percentage of immature A2B5+ OPCs. This was observed without significant change in total cell number and cytotoxicity, and was accompanied by an increase in BrdU-labeled A2B5+ and O4+ cells. FACS analysis confirmed a lack of apoptotic sub-G1 cells and revealed a greater percentage of S- and G2/M-phase OPCs with IFN-gamma treatment. Dual immunostaining with Ki-67 and Olig2 showed a smaller percentage of Olig2+ cells in G0 phase in IFN-gamma-treated OPCs, indicating loss of G1 control. Instead, increased levels and phosphorylation of the checkpoint protein p34cdc2 by IFN- suggested increased partial arrest in G2. IFN-gamma not only sustained expression of PCNA and the G1-S regulators retinoblastoma protein, cyclin D1, cyclin E, and cdk2, but also decreased p27 levels. In addition to changes in cell proliferation and differentiation, IFN-gamma attenuated myelin basic protein (MBP) expression significantly, which was associated with decreased expression of both MBP and Sox10 RNAs. These findings indicate that IFN-gamma not only maintains cell cycle activity that could predispose OPCs to apoptosis, but also overrides G1-G0 signals leading to thyroid hormone-mediated terminal differentiation and myelin gene expression.

Pulsatile Administration of the Epidermal Growth Factor Receptor Inhibitor Gefitinib Is Significantly More Effective than Continuous Dosing for Sensitizing Tumors to Paclitaxel

Gefitinib is an inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. Continuous inhibition of EGFR signaling is thought necessary for optimal inhibition of tumor cell proliferation. We hypothesized that continuous gefitinib may antagonize the effects of cytotoxics that inhibit tumor cells in other phases of the cell cycle. Furthermore, we hypothesized that intermittent dosing would allow for dose escalation and greater inhibition of EGFR-dependent antiapoptotic pathways. To test these assertions, we compared combinations of paclitaxel and gefitinib using either intermittent or continuous dosing schedules in mice. We found that when used in combination with paclitaxel, pulsatile gefitinib was significantly superior to continuous dosing. When gefitinib was administered for one or two consecutive days before paclitaxel, much higher doses could be given safely. Two days of gefitinib treatment before paclitaxel was most effective, causing significantly greater mean tumor regression and a higher percentage of complete responses than other schedules. The results suggest that the dose and schedule of an EGFR inhibitor required to effectively inhibit proliferation may differ from that required to stimulate apoptosis or to induce other effects. The former may require continuous EGFR inhibition to maintain cell cycle arrest, whereas sensitization to apoptosis may be optimally induced by profound but temporary inhibition of survival pathways. Our data suggest that the effects of receptor inhibition vary as a function of dose and schedule and that continuous administration of tyrosine kinase inhibitors may not be the best schedule with which to combine such agents with taxanes.