DNA Synthetic Phase Research Articles

A Mixture of Nucleosides and Nucleotides Increases Bone Marrow Cell and Peripheral Neutrophil Number in Mice Infected with Methicillin-Resistant Staphylococcus aureus

We studied the effects of a mixture of nucleosides and nucleotides on the peripheral neutrophil number and the proliferation of bone marrow cells in mice challenged with methicillin-resistant Staphylococcus aureus. BALB/c mice were fed a nucleotide-free 20% casein diet (control) or this diet supplemented with nucleosides and nucleotides orally (Expt. 1) or intraperitoneally (Expt. 2 and 3). On d 10, the mice were challenged intravenously with methicillin-resistant S. aureus (6.7 × 1012 colony forming units/L). In Expt. 1 and 2, numbers of total and differential counts of blood leucocytes were counted on d 0 (before), 1, 3 and 5 after the infection. In Expt. 3, 30 min before killing, bromodeoxyuridine (20 mg/kg), an analogue of thymidine, was administered intraperitoneally and its incorporation in the DNA synthetic phase of bone marrow cells was determined at 0 h (before), 3, 6 and 24 h after the infection. Mice fed the supplemented diet had higher (P < 0.05) leucocyte and neutrophil numbers on d 0 compared with the control group. The neutrophil numbers tended to be greater in the supplemented group at 1, 3 and 5 d after the infection. Intraperitoneal administration of nucleosides and nucleotides increased (P < 0.05) neutrophil numbers before and after the infection. Twenty-four h after the infection, incorporation of bromodeoxyuridine into the DNA synthetic phase of bone marrow cells in the group administered nucleosides and nucleotides was higher (P < 0.05) than in the control group. We conclude that, following methicillin-resistant S. aureus injection, intraperitoneal administration of a nucleoside-nucleotide mixture may stimulate bone marrow cell proliferation and increase the peripheral blood neutrophil numbers. Oral administration may elicit weaker effects.

Anticancer and Apoptotic-Inducing Effects of Rutin-Chitosan Nanoconjugates in Triple Negative Breast Cancer Cells

Cancer includes a range of diseases that arise as a result of the unregulated growth of malignant cells, which have the potential to invade or spread to other body parts. Nanoparticles and drug conjugate systems enable unique approaches for cancer treatment. The present study focused on anti-breast cancer activity of rutin-chitosan nanoconjugates in in vitro system. Rutin-chitosan nanoconjugates were prepared by ionic gelation process and characterized by UV–Vis Spectra analysis, DLS, SEM, Zeta potential, FT-IR and XRD analysis. Further the nanoconjugates was evaluated for its anti-cancer activity on Triple Negative Breast Cancer (TNBC), it shows significant activity against selected breast cancer cells. The Inhibitory concentration (IC50) of synthesized nano-conjugate was found to be 12.5 µg/mL. Furthermore, we found noteworthy cell cycle arrest at DNA synthetic phase following apoptotic cell deaths which were evidenced by AO/EtBr and DAPI fluorescence micrographic analysis. Our findings apparently validate that synthesized nano-conjugates could efficiently initiate the apoptotic process in the triple negative breast cancer cells. Through this finding, it is evidenced that the synthesized nanoconjugates potential applications in anti-cancer therapies.

Targeting CDK4 and CDK6: From Discovery to Therapy.

Biochemical and genetic characterization of D-type cyclins, their cyclin D-dependent kinases (CDK4 and CDK6), and the polypeptide CDK4/6 inhibitor p16(INK4)over two decades ago revealed how mammalian cells regulate entry into the DNA synthetic (S) phase of the cell-division cycle in a retinoblastoma protein-dependent manner. These investigations provided proof-of-principle that CDK4/6 inhibitors, particularly when combined with coinhibition of allied mitogen-dependent signal transduction pathways, might prove valuable in cancer therapy. FDA approval of the CDK4/6 inhibitor palbociclib used with the aromatase inhibitor letrozole for breast cancer treatment highlights long-sought success. The newest findings herald clinical trials targeting other cancers. Rapidly emerging data with selective inhibitors of CDK4/6 have validated these cell-cycle kinases as anticancer drug targets, corroborating longstanding preclinical predictions. This review addresses the discovery of these CDKs and their regulators, as well as translation of CDK4/6 biology to positive clinical outcomes and development of rational combinatorial therapies.

Bioluminescence Imaging of DNA Synthetic Phase of Cell Cycle in Living Animals

Bioluminescence reporter proteins have been widely used in the development of tools for monitoring biological events in living cells. Currently, some assays like flow cytometry analysis are available for studying DNA synthetic phase (S-phase) targeted anti-cancer drug activity in vitro; however, techniques for imaging of in vivo models remain limited. Cyclin A2 is known to promote S-phase entry in mammals. Its expression levels are low during G1-phase, but they increase at the onset of S-phase. Cyclin A2 is degraded during prometaphase by ubiquitin-dependent, proteasome-mediated proteolysis. In this study, we have developed a cyclin A2-luciferase (CYCA-Luc) fusion protein targeted for ubiquitin-proteasome dependent degradation, and have evaluated its utility in screening S-phase targeted anti-cancer drugs. Similar to endogenous cyclin A2, CYCA-Luc accumulates during S-phase and is degraded during G2/M-phase. Using Cdc20 siRNA we have demonstrated that Cdc20 can mediate CYCA-Luc degradation. Moreover, using noninvasive bioluminescent imaging, we demonstrated accumulation of CYCA-Luc in response to 10-hydroxycamptothecin (HCPT), an S-phase targeted anti-cancer drug, in human tumor cells in vivo and in vitro. Our results indicate that a CYCA-Luc fusion reporter system can be used to monitor S-phase of cell cycle, and evaluate pharmacological activity of anti-cancer drug HCPT in real time in vitro and in vivo, and is likely to provide an important tool for screening such drugs.

Fuel feeds function: Energy balance and bovine peripheral blood mononuclear cell activation

A general phenomenon in peripartum mammals is the breakdown of (acquired) immunity. The incidence of parasite load, disease and inflammation often rise during the specific energetically demanding time of pregnancy and lactation. In this period, blood leukocytes display decreased DNA synthesis in response to mitogens in vitro. Leukocyte activation, the phase of the cell cycle preceding the DNA synthetic phase has hardly been investigated, but the few studies suggest that leukocyte activation may also be impaired by the limited energy/nutrient availability. Leukocyte activation is characterized by manifold processes, thus, we used the cellular oxygen consumption rate (OCR) as a measure of ATP turnover to support all these processes. We hypothesized that the activation of peripheral blood mononuclear cells (PBMC) – in terms of oxygen consumed over basal levels after in vitro stimulation – is altered by energy balance around parturition. We studied peripartum high-yielding dairy cows because they undergo substantial fluctuations in energy intake, energy output and body fat mass. We established a fluorescence-based test strategy allowing for long-term (≥24h) quantification of O2-consumption and studied the peripartum period from 5weeks ante partum to 5weeks postpartum. In addition, we determined cellular lactate production, DNA/RNA synthesis and cell size and zoo-technical parameters such as animal energy intake and milk yield were assessed, as well as selected plasma parameters, e.g. glucose concentration. The basal OCR of PBMC from pregnant, non-lactating cows (n=6, −5weeks ante partum) was 1.19±0.15nmolmin−1 (107cells)−1 and increased to maximum levels of 2.54±0.49nmolmin−1 (107cells)−1 in phytohemagglutinin (PHA)-stimulated PBMC. The basal OCR did not change over the peripartum period. Whereas the activation indices, herein defined as the PHA-induced 24h-increase of OCR above baseline, amounted to 1.1±0.3, 4.2±0.3, 4.1±1.1, 2.1±0.3, and 2.7±0.5 at weeks −5, −1, +1, +2, and +5 relative to parturition, respectively. Because the activation index was positively correlated to plasma glucose levels and to energy balance during late pregnancy (week −5/week −1) and transition to lactation (week −1/week +2), we conclude that PBMC activation is modulated by energy/nutrient availability. In future studies, the activation index should aid the identification of causal mechanisms of disparity in PBMC activation, such as attenuated ion transport or macromolecule synthesis.

GENETIC VARIATION AND DNA REPLICATION TIMING, OR WHY IS THERE LATE REPLICATING DNA?

Mutation rates vary significantly within the genome and across species. Recent studies revealed a long suspected replication-timing effect on mutation rate, but the mechanisms that regulate the increase in mutation rate as the genome is replicated remain unclear. Evidence is emerging, however, that DNA repair systems, in general, are less efficient in late replicating heterochromatic regions compared to early replicating euchromatic regions of the genome. At the same time, mutation rates in both vertebrates and invertebrates have been shown to vary with generation time (GT). GT is correlated with genome size, which suggests a possible nucleotypic effect on species-specific mutation rates. These and other observations all converge on a role for DNA replication checkpoints in modulating generation times and mutation rates during the DNA synthetic phase (S phase) of the cell cycle. The following will examine the potential role of the intra-S checkpoint in regulating cell cycle times (GT) and mutation rates in eukaryotes. This article was published online on August 5, 2011. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected October 4, 2011.

Constitutively active RhoA inhibits proliferation by retarding G 1 to S phase cell cycle progression and impairing cytokinesis

The actions of RhoA in cytoskeletal regulation have been extensively studied. RhoA also contributes to proliferation and oncogenic transformation by less well-characterized means. Elevated RhoA signalling has been associated with human cancer; through increased RhoA expression, mutation or elevated expression of activating Rho guanine-nucleotide exchange factors (GEFs), or from deletion or decreased expression of inhibitory Rho GTPase-activating proteins (GAPs). Unlike the Ras oncogene, constitutively-activated GTPase-deficient RhoA mutants have not been identified in tumours. To investigate the effects of active RhoA on proliferation, we generated Swiss3T3 cells that inducibly express wild-type RhoA or GTPase-deficient active V14RhoA. We found that V14RhoA inhibited cell proliferation by retarding entry into the DNA synthetic cell cycle phase and blocking successful completion of cytokinesis, resulting in an increased incidence of binucleate cells. These effects were associated with inhibition of mitogen-induced activation of the MAPK pathway, and suppression of several proteins involved in mitosis, including anillin, ECT2 and cyclin B1 which would be expected to result in reduced activation of endogenous RhoA at the cell equator. Accumulation of active RhoA protein in the midbody of cells in telophase was inhibited in V14RhoA-expressing cells, suggesting that RhoA inactivation must occur prior to re-activation. Defective cytokinesis was also associated with prominent actin structures in V14RhoA-expressing cells, which might be incompatible with equatorial furrowing. Using super-resolution imaging based on single-molecule switching, we have significantly improved the resolution of active RhoA in midbodies. These results indicate that constitutively-active RhoA antagonizes several cellular activities that contribute to proliferation, highlighting the importance for cycling between GTP/GDP-bound states.

αII-Spectrin Is Critical for Cell Adhesion and Cell Cycle

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.

Characterization and localization of ORFF gene from the LD1 locus of Leishmania donovani

The Leishmania genome project has identified new genes at a rapid rate. The 32.8-megabase haploid genome of Leishmania major (Friedlin strain) is published and the comparative analysis of genome sequences of two other species, Leishmania infantum and Leishmanai braziliensis has been done. The haploid genome of Leishmania major (Friedlin strain) has around 8272 protein-coding genes, of which only 36% can be ascribed a putative function. Out of these open reading frames around 910 Leishmania major genes have no orthologs in the other two Tritryp genomes. These “ Leishmania -restricted” genes hold a potential as novel drug targets and potential vaccine candidates. Open reading frame, ORFF, is a single copy gene located on the chromosome 35 as a part of the multigene LD1 locus. Indirect immunofluorescence study and creation of ORFF-GFP fusion showed that ORFF is localized in the DNA containing compartments of Leishmania donovani, the nucleus and the kinetoplast. In order to characterize ORFF gene of L. donovani, we have created ORFF over-expressors and single allele deletion mutants by homologous replacement strategy. ORFF is likely to be an important gene for the parasite growth since results from over-expression studies and characterization of ORFF heterozygous knockout mutants reveal marked alterations in the cell cycle phenotype compared to the wild-type parasites. Flowcytometry based cell cycle analysis showed selective increase in the DNA synthetic phase of the ORFF over-expressors and a subversion of the same in heterozygous knockouts of ORFF suggesting its potential role in cell cycle progression.

Response of human mammary epithelial cells to DNA damage induced by 4-hydroxyequilenin: Lack of p53-mediated G 1 arrest

Long-term exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. One potential mechanism of estrogen carcinogenesis involves catechol formation and these catechols are further oxidized to electrophilic/redox active o-quinones, which have the potential to both initiate and promote the carcinogenic process. 4-Hydroxyequilenin (4-OHEN), a major phase I metabolite of several estrogens present in Premarin ®, is considerably more cytotoxic, carcinogenic, and mutagenic as compared to the catechol estrogen metabolites of endogenous estrogens. Previously, we showed that 4-OHEN autoxidized to an o-quinone and caused a variety of damage to DNA. Allowing more time between the induction of DNA damage and the entry of a damaged cell into the DNA synthetic phase of the cell cycle protects that cell from mutagenesis. Central to this response is the establishment of a G 1 checkpoint. This checkpoint is mediated by the cyclin-dependent kinase inhibitor p21 WAF1, a direct downstream target for transcriptional activation by p53. In this study, we investigated this signaling pathway. Surprisingly, exposure of the human MCF-10A immortalized nontransformed mammary epithelial cell line to 4-OHEN did not induce a p53-induced G 1 arrest. A 24 h treatment with 4-OHEN significantly induced p53 and p21 WAF1 protein expression at 10 and 20 μM, as well as significantly induced the transactivation of a p53-luciferase reporter gene at 20 μM. Significant decreases in cell proliferation were also observed with concentrations of 5 μM and higher of 4-OHEN. However, 4-OHEN did not induce a G 1 checkpoint and cells with damaged DNA accumulated in the S phase. This S phase delay could be beneficial for the survival of the damaged cells which could contribute to the carcinogenic process.

G1 Phase Regulation, Area-Specific Cell Cycle Control, and Cytoarchitectonics in the Primate Cortex

We have investigated the cell cycle-related mechanisms that lead to the emergence of primate areas 17 and 18. These areas are characterized by striking differences in cytoarchitectonics and neuron number. We show in vivo that (1) area 17 precursors of supragranular neurons exhibit a shorter cell cycle duration, a reduced G1 phase, and a higher rate of cell cycle reentry than area 18 precursors; (2) area 17 and area 18 precursors show contrasting and specific levels of expression of cyclin E (high in area 17, low in area 18) and p27Kip1 (low in area 17, high in area 18); (3) ex vivo up- and downmodulation of cyclin E and p27Kip1 show that both regulators influence cell cycle kinetics by modifying rates of cell cycle progression and cell cycle reentry; (4) modeling the areal differences in cell cycle parameters suggests that they contribute to areal differences in numbers of precursors and neuron production.

High flow drives vascular endothelial cell proliferation during flow-induced arterial remodeling associated with the expression of vascular endothelial growth factor

Endothelial cell activation and proliferation are the essential steps in flow-induced arterial remodeling. We investigated endothelial cell turnover in the early stages of high-flow in the rabbit common carotid arteries using an arteriovenous fistula (AVF) model by kinetic investigation of cell proliferation and cell molecular analysis. BrdU was administrated to label endothelial cells (ECs) in DNA synthetic phase (S-phase) of the cell mitotic cycle. Pulse labeling revealed that ECs entered S-phase at 1.5 days of AVF (0.93 ± 0.19%). Endothelial cell labeling index (EC-LI) peaked at 2 days of AVF (8.90 ± 0.87%) with a high index of endothelial cell mitosis (EC-MI, 1.67 ± 0.47%). Endothelial cell density increased remarkably at 3 days of AVF with a significant decrease in EC-LI (54%) and EC-MI (60%). Study of kinetics of EC proliferation revealed that endothelial cells took 16–24 h to finish one cycle of cell mitosis. Tracking investigation of pulse BrdU-labeled endothelial cells at 1.5 days showed that more than 66% of endothelial cells were BrdU-labeled 1.5 days after labeling. VEGF, integrin ανβ3, PECAM-1, and VE-cadherin were upregulated significantly preceding endothelial cell proliferation and kept at high levels during endothelial cell proliferation. These data suggest that endothelial cell proliferation is the initial step in flow-induced arterial remodeling. Hemodynamic forces may drive endothelial cell downstream migration. Expression of VEGF and cell junction molecules contribute to flow-induced arterial remodeling.

Using the male gamete for assisted reproduction: past, present, and future.

Using the male gamete for assisted reproduction: past, present, and future.

EFFECT OF N-PROOPIOMELANOCORTIN (1-77) AND (1-49) INFUSIONS ON ADRENAL EXPRESSION OF CYCLIN D1 IN THE FETAL SHEEP

In the sheep, there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10–15 days gestation. Recently, we have shown that infusion of POMC 1–77 increases fetal adrenal growth but does not significantly alter fetal plasma cortisol concentrations. Phosphorylation and inactivation of the pRB protein, which is required for progression into the DNA synthetic phase of the cell-cycle is conducted by a holoenzyme, for which cyclin D1 gene encodes the rate-limiting regulatory subunit. To further elucidate the mechanisms by which POMC 1–77 regulates adrenal growth, we therefore examined adrenal expression of the rate-limiting cell cycle protein, cyclin D1, from fetuses infused for 48 hr with POMC 1–77 (n = 6), POMC 1–49 or Saline (n = 6). There was no significant difference in the adrenal expression of cyclin D1 mRNA levels between POMC 1–77, 1–49 and saline infused fetuses. There was no significant correlation between cyclin D1 (4.0 Kb) and adrenal weight. In summary, these data do not demonstrate that the rate-limiting cell cycle protein, cyclin D1, is activated to stimulate adrenal growth following infusion of POMC 1–77 in the fetal sheep in late gestation.

DNA cytofluorometric analysis of chondrocytes in human articular cartilages under normal aging or arthritic conditions

Objective Since most chondrocytes in articular cartilage are in the resting phase (G0) of the cell cycle, it has been difficult to investigate their cell kinetics using 3H-thymidine autoradiography, or immunohistochemistry. In the present study, DNA cytofluorometry, which is useful to analyse the cell kinetics even for such inactive cell populations as in the G0 phase, was applied to human chondrocytes of the articular cartilages under normal aging and pathologic conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), and aseptic necrosis (AN).Design The human articular cartilages for the study were obtained from autopsy and surgical materials. Fifty joints were used for the study of aging, 54 for the study of OA, 20 for studying RA, and 10 for AN study. The isolated chondrocytes were quickly prepared from fresh articular cartilages, using a combination method of enzymatic digestion with papain and collagenase, followed by mechanical cell separation by churning and homogenization.Results The DNA histograms obtained by cytofluorometry with propidium-iodide staining showed that most chondrocytes had diploid DNA content (2c) in all cartilages studied, suggesting that they were in the G0 phase. However, there were a few chondrocytes having tetraploid DNA content (4c) in the normally aged articular cartilages, and there were some cells having DNA content between 2c and 4c in the diseased cartilages. The former cells were considered to be G0-phase cells of the 4c chondrocytes, while the latter cells were considered to be in the DNA synthetic (S) phase or G2-phase of the 2c chondrocytes. The frequency of 4c chondrocytes in aged cartilage was significantly increased, compared to that in the young cartilage. In contrast to the normal cartilage, the frequency of S- and G2-phase cells, which was expressed as the S– G2 index, in diseased cartilages (OA, RA and AN) was significantly high (P< 0.0001). In OA cartilage, the S–G2 index was much higher in the severe or moderate stage than in the mild stage, suggesting that the chondrocytes in clusters may actively proliferate.Conclusion These results showed that in normal articular cartilages most chondrocytes are in the G0 phase, while some became 4c polyploid cells, and that these G0-phase chondrocytes had a potential to proliferate under diseased conditions.

Development of astrocytes in the mouse embryonic cerebrum tracked by tenascin-C gene expression.

The development of astrocytes in the rodent cerebrum has been considered to occur mainly in the perinatal stage after the end of neurogenesis, and the mode of generation and migration of the astroglial precursors in the embryonic cerebrum remains poorly understood. Tenascin-C (TN-C) is an astroglia-derived extracellular matrix molecule, and its expression can be traced to the ventricular germinative zone of the embryonic cerebrum. TN-C expression may therefore be used as a marker of astroglial precursors prior to the expression of the glial fibrillary acidic protein (GFAP), a marker for the late stage of astroglial differentiation. The development of astrocytes in the mouse embryonic cerebrum was examined by in situ hybridization histochemistry for TN-C mRNA as an early marker. Observations showed that TN-C-positive cells were aligned in the ventricular germinative zone and began to migrate radially at E (embryonic day) 15. They were dispersed into the cortex as early as E17, and some were still in the DNA synthetic phase (S phase) even after lodgement in the cortex. TN-C-positive cells differentiated into GFAP-immunopositive astrocytes in the cortical and subcortical areas at the neonatal stage. The distribution of their active generation sites in the ventricular germinative zone was not homogeneous but displayed a restricted pattern. The mode of their migration towards the cortical and subcortical areas also showed a characteristic pattern that correlated to the radial glial arrangement. These findings indicate that TN-C-positive cells represent a new subset of astroglial precursors which arise directly from the ventricular germinative zone, and that astrogliogenesis from this precursor occurs as early as the period of neurogenesis at the embryonic stage.

Growth State-Dependent Binding of USF-1 to a Proximal Promoter E Box Element in the Rat Plasminogen Activator Inhibitor Type 1 Gene

Induced PAI-1 gene expression in renal epithelial (NRK-52E, clone EC-1) cells occurs as part of the immediate-early response to serum. PAI-1 transcripts are maximally expressed early in G1 (within 4 h of serum addition to quiescent EC-1 cells) and then subsequently decline to basal levels prior to entry into DNA synthetic phase. Comparative analysis of PAI-1 mRNA abundance and de novo-synthesized thiolated RNA in quiescent cells, as well as at 4 h (early G1) and 20 h (late G2) postserum addition, in conjunction with RNA decay measurements indicated that PAI-1 gene regulation upon growth activation was predominantly transcriptional. An E box motif (CACGTG), important in the induced expression of some growth state-dependent genes, mapped to nucleotides −160 to −165 upstream of the transcription start site in the PAI-1 proximal promoter. Mobility-shift assessments, using a 18-bp deoxyoligonucleotide construct containing the E box within the context of PAI-1-specific flanking sequences, confirmed binding of EC-1 nuclear protein(s) to this probe and, specifically, to the E box hexanucleotide site. The specificity of this protein-probe interaction was verified by competition analyses with double-stranded DNA constructs that included E box deoxyoligonucleotides with non-PAI-1 flanking bases, mutant E box sequences incapable of binding NRK nuclear proteins, and unrelated (i.e., AP-1) target motifs. Extract immunodepletion and supershift/complex-blocking experiments identified one PAI-1 E box-binding protein to be upstream stimulatory factor-1 (USF-1), a member of the HLH family of transcription factors. Mutation of the CACGTG site to TCCGTG in an 18-bp PAI-1 probe inhibited the formation of USF-1-containing complexes confirming that an intact E box motif at −160 to −165 bp in the PAI-1 promoter and, in particular, the CA residues at −165 and −164 are essential for USF-1 binding. Incorporation of this 2 bp change into a reporter construct containing 764 bp of the proximal PAI-1 “promoter” ligated to a CAT gene effectively reduced (by 74%) CAT activity in cycling cells. An intact E box motif at nucleotides −160 to −165 in the PAI-1 promoter, thus, is an important functional element in the regulation of PAI-1 transcriptional activity in renal cells.

Wnt1 and MEK1 Cooperate to Promote Cyclin D1 Accumulation and Cellular Transformation

Members of the Wnt family of signal transducers regulate cellular differentiation/reorganization and cellular proliferation. However, few pro-proliferative targets of Wnt have been identified. We now show that cyclin D1, a critical mediator of cell cycle progression, is a downstream target of Wnt-dependent signaling. NIH-3T3 cell lines engineered to overexpress Wnt1 displayed reduced glycogen synthase kinase-3beta activity. Wnt1-dependent glycogen synthase kinase-3beta inhibition corresponded with decreased cyclin D1 proteolysis and, thus, hyperaccumulation of active cyclin D1.CDK4 (cyclin-dependent kinase 4) kinase. However, in the absence of serum-derived growth factors, Wnt-1 was not sufficient to drive cyclin D1 accumulation or S-phase entry. In contrast, cells engineered to co-express Wnt1 and activated MEK1 accumulated high levels of cyclin D1 and entered the DNA synthetic phase in the absence of serum-derived growth factors, a characteristic of neoplastic transformation. The ability of a dominant-negative cyclin D1 mutant, D1-T156A, to inhibit Wnt1/MEK1-dependent S-phase entry suggests that cyclin D1 is a critical downstream target for Wnt1- and MEK1-dependent cellular proliferation.

INK4d-deficient mice are fertile despite testicular atrophy.

The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.

The INK4 family of cell cycle inhibitors in cancer.

Since the discovery of p16 (Serrano et al., 1993) and p21 (El-Deiry et al., 1993; Harper et al., 1993; Xiong et al., 1993), the cyclin-dependent kinase inhibitors (CKIs) have been intensively investigated. CKIs include two distinct families, the INK4 (INhibitors of CDK4) family, whose four members (p16, p15, p18, p19) exclusively bind to and inhibit the D-type cyclin-dependent kinases (CDK4 and CDK6), and the CIP/KIP family, whose three members (p21, p27, p57) are able to inhibit the activity of all CDKs (Sherr and Roberts, 1995). The roles of these cell cycle inhibitors are being elucidated through studies of mouse knock-out models, gene expression in cell lines, and genetic screens of human tumors. Enforced expression of any of the CKIs results in growth arrest in the G1 phase and prevents entry into the DNA synthetic (S) phase (Sherr and Roberts, 1995). Growth arrest is accomplished by inhibiting retinoblastoma protein (pRB) phosphorylation (Ewen, 1994), and thus the release of the E2F transcription factors, which are required for the transcriptional activation of genes necessary for DNA synthesis (Dyson, 1998; Nevins, 1998). These similar biological and biochemical properties suggested that CKIs might act as tumor suppressors and that deletion, mutation, or transcriptional repression of the CKI genes would lead to uncontrolled cell growth and cancer. Screens of human tumors for genetic alterations (including mutations, deletions, rearrangements, or promoter hypermethylation and silencing), and the creation and analyses of knock-out mice, clearly implicate the INK4a/ARF locus, as an important target in cancer. In contrast, the other INK4 or CIP/ KIP loci do not appear to be involved in the etiology of cancer (Hirama and Koe‚er, 1995; Hall and Peters, 1996; Drexler, 1998). Several excellent reviews on CKIs (Harper and Elledge, 1996; Nakayama and Nakayama, 1998) and the roles of p16 (Ruas and Peters, 1998) and the INK4a/ARF locus in cancer have been recently published (Sherr, 1998; Sharpless and DePinho, 1999). Therefore, here, I will review only the most recent data (from our institution and from others), on the alterations of the INK4 genes in human tumors, and the functional signi®cance of targeted deletions of INK4 genes in the mouse, alone or in combination with the deletions of other CKIs. The INK4 family: chromosomal location and structure