Activity Of Cell Cycle Regulators Research Articles

Abstract LB191: A genome-wide CRISPR-based functional screen uncovers the KLF4-Survivin/BIRC5 signaling pathways as a key regulator of cell fate following endoplasmic reticulum stress: Implications for tumor growth and therapeutic targeting

Abstract The growth and spread of tumors involve intricate interactions with the surrounding tissue microenvironment. As a tumor develops, it faces various stresses from its microenvironment, including hypoxia, nutrient deficiency, and acidosis. In response to these challenges, cancer cells can hijack existing cytoprotective mechanisms, such as the Unfolded Protein Response (UPR). The UPR activates signaling pathways at the translational and later transcriptional levels to alleviate cellular stress and prevent cell death. However, under unresolved or chronic endoplasmic reticulum (ER) stress, the UPR may paradoxically promote cell death. The precise mechanisms through which the UPR influences cellular fate during ER stress remain poorly understood. To address this knowledge gap, we employed a functional CRISPR-based genetic knockout screen to identify novel regulators of the UPR. Using a lentiviral genome-wide CRISPR-Cas9 knockout library, we identified over-represented sgRNAs (associated with pro-apoptotic genes) and underrepresented sgRNAs (associated with pro-survival genes) following ER stress induced by thapsigargin and tunicamycin. One of the top candidates identified was Survivin/BIRC5, a protein with anti-apoptotic and cell cycle regulatory activities and which is overexpressed in tumor cells compared to healthy tissues. Functional studies revealed that genetic or pharmacological inhibition of Survivin expression enhanced ER stress-induced apoptosis, but not apoptosis in response to other forms of stress. These findings were validated in multiple cancer cell lines, ruling out off-target effects. Mechanistically, ER stress led to PERK-dependent downregulation of Survivin, and overexpression of Survivin blocked Thapsigargin-induced apoptosis. In vivo xenograft models showed that combining Survivin ablation with PERK inhibition significantly delayed tumor growth and improved overall survival. Through a combination of bioinformatics analysis and loss-of-function studies, we identified the transcription factor KLF4 as a mediator of PERK-dependent effects on Survivin repression. Notably, CRISPR-mediated KLF4 knockout successfully rescued Survivin levels post-ER stress. Additionally, an analysis of TCGA data across various tumor types provided supporting evidence of a negative correlation between KLF4 and Survivin expression. In summary, this study unveils a novel regulatory axis, PERK-KLF4-Survivin, as a key determinant of cell fate in the cellular responses to ER stress. Targeting Survivin in conjunction with PERK inhibition showed promising antitumor effects in vivo. Citation Format: Nektaria M. Leli, Hengxi Liu, Souvik Dey, Lauren Brady, Carlo Salas Salinas, Victoria Wu, Spiros Tastsoglou, Giorgos Skoufos, Ioannis Verginadis, Ilias Karagounis, Artemis G. Hatzigeorgiou, Constantinos Koumenis. A genome-wide CRISPR-based functional screen uncovers the KLF4-Survivin/BIRC5 signaling pathways as a key regulator of cell fate following endoplasmic reticulum stress: Implications for tumor growth and therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB191.

Pentagamavunone-1 inhibits aggressive breast cancer cell proliferation through mitotic catastrophe and ROS-mediated activities: in vitro and in vivo studies

Pentagamavunone-1 (PGV-1), an analog of curcumin, has been studied for its cytotoxic effects in 4T1, MCF7, MCF7/HER2, and T47D breast cancer cells. Its antiproliferative effect is partly mediated through G2/M arrest; however, its molecular mechanism during cell cycle progression remains unknown. In this study, we aimed to determine whether PGV-1 has any anticancer effects on highly aggressive breast cancer cells, with a focus on cell cycle regulatory activity, reactive oxygen species (ROS) generation, and their mediated effects on cancer cells. MDA-MB-231 (triple-negative) and HCC1954 (overexpressed HER2) immortalized human breast cancer cells were used in the study. PGV-1 exhibited cytotoxic activity with an irreversible antiproliferative impact on treated cells and had good selectivity when tested in fibroblast cells. Oral PGV-1 administration suppressed tumor growth in a cell-derived xenograft mouse model. PGV-1 induced the phosphorylation of Aurora A kinase and PLK1 in MDA-MB-231 cells, while PLK1 and cyclin B1 phosphorylation were enhanced in the PGV-1-treated HCC1954 cells during prometaphase arrest. Intracellular ROS production was substantially higher upon PGV-1 treatment following mitotic arrest, and this activity caused impairment of mitochondrial respiration, induced senescence, and subsequently triggered early-to-late apoptosis. Collectively, these results suggest that the molecular mechanism of PGV-1 involves the regulation of mitotic kinases to cause cell cycle arrest and the enhancement of ROS production to impair mitochondrial activity and induce cellular senescence. The therapeutic activities demonstrated by PGV-1 in this study show its potential as an appealing candidate for chemotherapy in breast cancer treatment.

Abstract P1029: Improving Cardiomyocyte Cell Cycle Entry In Response To Direct Cell Cycle Stimulation Using A Novel Small Molecule Inhibitor Of P38

Background: Myocardial infarction is the leading cause of mortality globally, due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we showed that overexpression of a combination of 4 cell cycle factors, CDK1, CDK4, cyclin B1, and cyclin D1 (collectively known as 4F), induced cell division in ~15% of post-mitotic mouse, rat, and human CMs. The aim of the current study is to identify novel small molecules that further augment CM cycle induction caused by the 4F. Methods and Results: We performed a drug screening of 30 chemical compounds with possible cell cycle regulatory activities using 60-day-old mature hiPS-CMs overexpressing the 4F and assessed the increase in EDU (DNA synthesis marker) and PHH3 (G2-M phase marker). The top hit was N-(4,6-Dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide (NDPPC). NDPPC showed a dose-dependent increase in the percentage of EDU and PHH3 positive nuclei in hiPS-CMs transduced with 4F. CMs transduced with 4F and treated with 1 μM NDPPC showed a significant increase in the percentage of EDU and PHH3 compared with vehicle (V)-treated CMs (PHH3; V: 20.41±2.02%, NDPPC: 33.75±3.9%, EDU; V: 18.24±1.7%, NDPPC: 30.81±2.1% n=6, p<0.001). In Silico Drug-Target prediction showed that NDPPC could interact with p38 mitogen-activated protein kinase (P38), a critical negative regulator of mammalian cell cycle. Therefore, we overexpressed P38, in the presence and absence of NDPPC and 4F. As expected, overexpression of P38 in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect (in presence of 4F PHH3%; V: 20.38±0.97%, P38 overexpression+V: 11.02±1.3%, P38 overexpression+NDPPC: 23.62±2.79, n=4, P<0.001). Knockdown of P38 showed a significant increase in the percentage of EDU and PHH3 positive nuclei in 4F treated CMs, and treatment with NDPPC did not show any further increase in cell cycle induction. These data indicate that NDPPC enhances the 4F cell cycle response in CMs through inhibition of P38. Conclusions: NDPPC is a novel inhibitor for P38. NDPPC is a promising drug to promote CM cell cycle response to the 4F. Further work in vivo is needed to test whether NDPPC could improve the 4F effect on cardiac function after ischemic injury.

Suppression of centrosome protein TACC3 induces G1 arrest and cell death through activation of p38–p53–p21 stress signaling pathway

The centrosome regulates diverse cellular processes, including cell proliferation and differentiation. TACC3, a member of the human transforming acidic coiled-coil protein family, is a key centrosomal protein that is up-regulated in many cancers. Previous studies have demonstrated that TACC3 is essential for the survival of vertebrates and is involved in cell cycle regulation in human cells. However, the details of the underlying mechanisms in its cell cycle regulatory activity remain poorly understood. In this study, we showed that suppression of TACC3 expression induced G1 cell cycle arrest and triggered cell death in human cells. TACC3 depletion-induced G1 arrest and cell death were significantly reduced in cells either lacking p53 or with pharmacologically-inhibited p38, indicating that G1 arrest and cell death induction both require p53 and p38. TACC3 depletion up-regulated the levels of p53 and p21 and induced the accumulation of p53 both in the nucleus and at the centrosome. Interestingly, TACC3 depletion led to the activation of p38 and stimulated the recruitment of activated p38 to the centrosome. Depletion of TACC3 up-regulated the phosphorylation of p53 at Serine 33, a site known to be phosphorylated by p38 under cellular stress and further induced the accumulation of phosphorylated p53 to the centrosome. Loss of TACC3 affected centrosome integrity by disrupting the localization of components of the γ-tubulin ring complex at the centrosome. The results demonstrate that TACC3 depletion induces G1 arrest and cell death by activating p38–p53–p21 signaling and triggering a centrosome-mediated cellular stress response.

Division of labour between Myc and G1 cyclins in cell cycle commitment and pace control.

A body of evidence has shown that the control of E2F transcription factor activity is critical for determining cell cycle entry and cell proliferation. However, an understanding of the precise determinants of this control, including the role of other cell-cycle regulatory activities, has not been clearly defined. Here, recognizing that the contributions of individual regulatory components could be masked by heterogeneity in populations of cells, we model the potential roles of individual components together with the use of an integrated system to follow E2F dynamics at the single-cell level and in real time. These analyses reveal that crossing a threshold amplitude of E2F accumulation determines cell cycle commitment. Importantly, we find that Myc is critical in modulating the amplitude, whereas cyclin D/E activities have little effect on amplitude but do contribute to the modulation of duration of E2F activation, thereby affecting the pace of cell cycle progression.

The antileukaemic cell cycle regulatory activities of swainsonine purified from Metarhizium anisopliae fermentation broth

Swainsonine is a Metarhizium secondary metabolite known differentially for its specific mannosidase inhibitory, toxic and therapeutic activities. Here, the standard and purified swainsonine from Metarhizium anisopliae fermentation broth were comparatively evaluated for their in situ antileukaemic activities in human promyelocytic cell line, HL-60. Both the standard (IC50 = 6.96 μM) and purified (IC50 = 9.50 μM) compounds inhibited the leukaemic cell proliferation without inflicting cell membrane disruption at 48 h of post-treatment incubation. The DNA cell cycle analysis showed approximately 48.81% and 60.72% of the treated cells arrested in the synthetic phase (S-phase) at 36 and 48 h, respectively, upon treatment with IC50 concentration of the purified swainsonine. However, only 29.62% of cells were arrested in S-phase with standard swainsonine at 48 h, suggesting the comprehensive action of certain other metabolites sharing the similar paradigm of antiproliferative properties in Metarhizium broth extract.

Daidzein promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and improves glucose homeostasis in Type 2 diabetic model mice

Daidzein shows estrogenic, antioxidant and antiandrogenic properties as well as cell cycle regulatory activity. However, the antihyperglycemic effect of daidzein remains to be elucidated. In this study, we investigated the in vitro effect of daidzein on glucose uptake, AMPK phosphorylation and GLUT4 translocation on plasma membrane in L6 myotubes and its in vivo antihyperglycmic effect in obese–diabetic model db/db mice. Daidzein was found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by daidzein of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a GLUT4 cDNA-coding vector. Daidzein (0.1% in the diet) suppressed the rises in the fasting blood glucose, serum total cholesterol levels and homeostasis model assessment index of db/db mice. In addition, daidzein supplementation markedly improved the AMPK phosphorylation in gastrocnemius muscle of db/db mice. Daidzein also suppressed increases in blood glucose levels and urinary glucose excretion in KK-Ay mice, another Type 2 diabetic animal model. These in vitro and in vivo findings suggest that daidzein is preventive for Type 2 diabetes and an antidiabetic phytochemical.

Evidence for a mammalian late-G1 phase inhibitor of replication licensing distinct from geminin or Cdk activity

Pre-replication complexes (pre-RCs) are assembled onto DNA during late mitosis and G1 to license replication origins for use in S phase. In order to prevent re-replication of DNA, licensing must be completely shutdown prior to entry into S phase. While mechanisms preventing re-replication during S phase and mitosis have been elucidated, the means by which cells first prevent licensing during late G1 phase are poorly understood. We have employed a hybrid mammalian / Xenopus egg extract replication system to dissect activities that inhibit replication licensing at different stages of the cell cycle in Chinese Hamster Ovary (CHO) cells. We find that soluble extracts from mitotic cells inhibit licensing through a combination of geminin and Cdk activities, while extracts from S-phase cells inhibit licensing predominantly through geminin alone. Surprisingly however, geminin did not accumulate until after cells enter S phase. Unlike extracts from cells in early G1 phase, extracts from late G1 phase and early S phase cells contained an inhibitor of licensing that could not be accounted for by either geminin or Cdk. Moreover, inhibiting cyclin and geminin protein synthesis or inhibiting Cdk activity early in G1 phase did not prevent the appearance of inhibitory activity. These results suggest that a soluble inhibitor of replication licensing appears prior to entry into S phase that is distinct from either geminin or Cdk activity. Our hybrid system should permit the identification of this and other novel cell cycle regulatory activities.

C-ETS1 Facilitates G1/S-phase Transition by Up-regulating Cyclin E and CDK2 Genes and Cooperates with Hepatitis B Virus X Protein for Their Deregulation

Recent studies on the molecular mechanisms responsible for cell cycle deregulation in cancer have puzzled out the role of oncogenes in mediating unscheduled cellular proliferation. This is reminiscence of their activity as proto-oncogenes that drives scheduled cell cycle progression under physiological conditions. Working on the cell cycle regulatory activity of proto-oncogene, we observed that c-ETS1 transcriptionally up-regulated both cyclin E and CDK2 genes, the master regulators of G(1)/S-phase transition. The process was mediated by kinetic coherence of c-ETS1 expression and its recruitment to both promoters during G(1)/S-phase transition. Furthermore, enforced expression of c-ETS1 helped G(0)-arrested cells to progress into G(1)/S-phases apparently due to the activation of cyclin E/CDK2 genes. Physiological induction of c-ETS1 by EGF showed the remodeling of mononucleosomes bound to the c-ETS1 binding site on both promoters during their activation. The exchange of HDAC1 with histone acetyltransferase-p300 was contemporaneous to the chromatin remodeling with consequent increase in histone H3K9 acetylation. Furthermore, the ATP-dependent chromatin remodeler hBRM1 recruitment was also associated with nucleosome remodeling and promoter occupancy of phospho-Ser5 RNA polymerase II. Intriguingly, the activity of the HBx viral oncoprotein was dependent on c-ETS1 in a hepatotropic manner, which led to the activation of cyclin E/CDK2 genes. Thus, cyclin E and CDK2 genes are key physiological effectors of the c-ETS1 proto-oncogene. Furthermore, c-ETS1 is indispensable for the hepatotropic action of HBx in cell cycle deregulation.

P38 MAPK and JNK Antagonistically Control Senescence and Cytoplasmic p16INK4A Expression in Doxorubicin-Treated Endothelial Progenitor Cells

Patients treated with low-dose anthracyclines often show late onset cardiotoxicity. Recent studies suggest that this form of cardiotoxicity is the result of a progenitor cell disease. In this study we demonstrate that Cord Blood Endothelial Progenitor Cells (EPCs) exposed to low, sub-apoptotic doses of doxorubicin show a senescence phenotype characterized by increased SA-b-gal activity, decreased TRF2 and chromosomal abnormalities, enlarged cell shape, and disarrangement of F-actin stress fibers accompanied by impaired migratory ability. P16 INK4A localizes in the cytoplasm of doxorubicin-induced senescent EPCs and not in the nucleus as is the case in EPCs rendered senescent by different stimuli. This localization together with the presence of an arrest in G2, and not at the G1 phase boundary, which is what usually occurs in response to the cell cycle regulatory activity of p16INK4A, suggests that doxorubicin-induced p16 INK4A does not regulate the cell cycle, even though its increase is closely associated with senescence. The effects of doxorubicin are the result of the activation of MAPKs p38 and JNK which act antagonistically. JNK attenuates the senescence, p16 INK4A expression and cytoskeleton remodeling that are induced by activated p38. We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity. In conclusion, this study provides a better understanding of the senescence of doxorubicin-treated EPCs, which may be helpful in preventing and treating late onset cardiotoxicity.

E2F1 in gliomas: A paradigm of oncogene addiction

Cancer arises as a result of a stepwise accumulation of genetic changes. One of these changes, deregulation of the Rb/E2F1 pathway resulting from alterations in members of the pathway, is a hallmark of all human cancers. These mutations promote tumor development by deregulating the E2F family of transcription factors, which results in uncontrolled cell cycle progression. The E2F1 protein functions as a transcription factor that enhances cell proliferation by binding to the promoter region of several genes, including those that are involved in cell cycle regulatory activities and DNA replication. It is now becoming clear that the role of E2F1 in regulating transcription and cell growth is also highly dependent on the cellular context. This complexity is also evident from analyses of perturbations in E2F-modulated tumor development. For example, deregulated E2F1 expression can either promote or inhibit tumorigenesis depending on the nature of the other oncogenic mutations that are present. This explains the ability of E2F1 to behave as both an oncogene and tumor suppressor gene. Here we focus on reviewing the most recent evidence supporting the “addiction” of gliomas to this versatile transcription factor. We also consider the clinical relevance of this by examining the role of E2F1 as a prognosis factor and as a target for the development of novel strategies.

Species Selectivity of Mixed-Lineage Leukemia/Trithorax and HCF Proteolytic Maturation Pathways

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.

PCAF Modulates PTEN Activity

The PTEN protein has a single catalytic domain possessing both lipid phosphoinositol and protein phosphatase activities. The lipid phosphoinositol phosphatase activity is essential for PTEN to block the cell cycle in the G1 phase and thereby to suppress tumor formation and progression (Cantley, L. C., and Neel, B. G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4240-4245), although the mechanisms governing PTEN activity under normal and neoplastic growth conditions remain unclear. Here, we report that PTEN interacts physically and functionally with PCAF, a histone acetyltransferase that regulates gene transcription through interaction with p300/CBP and various sequence-specific transcription factors (Nakatani, Y. (2001) Genes Cells 6, 79-86). Expression of PCAF results in increased acetylation of lysine residues (Lys125 and Lys128) within the catalytic cleft of PTEN, a structure essential for phosphatidylinositol 3,4,5-trisphosphate specificity (Lee, J. O., Yang, H., Georgescu, M. M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J. E., Pandolfi, P., and Pavletich, N. P. (1999) Cell 99, 323-334). The acetylation of PTEN caused by PCAF expression depends on the presence of growth factors. Reduction of endogenous PCAF activity using shRNA results in a loss of PTEN acetylation in response to growth factors and restores the ability of PTEN to down-regulate phosphatidylinositol 3-kinase signaling and to induce G1 cell cycle arrest. The retention of phosphatidylinositol 3-kinase/AKT signaling and cell cycle regulatory activities of acetylation-resistant PTEN K125R and K128R mutants in the presence of enforced PCAF expression suggest a causal relationship. Together, these findings indicate a mechanism of PTEN regulation that forges a link between distinct cancer-relevant pathways central to the control of growth factor signaling and gene expression.

Androgens target prohibitin to regulate proliferation of prostate cancer cells.

Proteins involved in the growth response of prostate cancer cells to androgen were investigated by comparing the proteomes of LNCaP cells treated with vehicle or androgen. Whole-cell lysates were separated by two-dimensional PAGE, and HPLC-MS/MS was used to identify androgen-regulated proteins. Prohibitin, a protein with cell-cycle regulatory activity, was shown to be downregulated by 50% following androgen stimulation. Western blot and reverse transcription-PCR experiments confirmed the result and showed that regulation occurs at the level of transcription. To determine the importance of prohibitin in androgen-stimulated growth, we used transient transfection to overexpress the protein and RNA interference to knock down the protein. Subsequent FACS analysis showed that cells with reduced levels of prohibitin showed a slight but reproducible increase in the percentage of population in cell cycle, while cells with increased prohibitin levels showed a clear reduction in the percentage entering cell cycle, following dihydrotestosterone stimulation, when compared to untransfected controls. Confocal microscopy showed localization of prohibitin in the nucleus as well as the mitochondria of LNCaP cells. It therefore seems that the regulation of prohibitin is a vital part of the cellular growth response to androgen stimulation in LNCaPs and prohibitin may have a nuclear regulatory role in cell-cycle progression.

The role of E2F4 in adipogenesis is independent of its cell cycle regulatory activity.

The E2F and pocket protein families are known to play an important role in the regulation of both cellular proliferation and terminal differentiation. In this study, we have used compound E2F and pocket protein mutant mouse embryonic fibroblasts to dissect the role of these proteins in adipogenesis. This analysis shows that loss of E2F4 allows cells to undergo spontaneous differentiation. The ability of E2F4 to prevent adipogenesis seems to be quite distinct from the known properties of E2F. First, it can be separated from any change in either E2F-responsive gene expression or cell cycle regulation. Second, it is a specific property of E2F4, and not other E2Fs, and it occurs independently of E2F4's ability to interact with pocket proteins. In addition, E2F4 loss does not override the differentiation defect resulting from pRB loss even though it completely suppresses the proliferation defect of Rb(-/-) mouse embryonic fibroblasts. This finding definitively separates the known, positive role of pRB in adipogenesis from its cell cycle function and shows that this pocket protein is required to act downstream of E2F4 in the differentiation process.

Functional analysis of the p57KIP2 gene mutation in Beckwith-Wiedemann syndrome.

p57KIP2 is a potent tight-binding inhibitor of several G1 cyclin/cyclin-dependent kinase (Cdk) complexes, and is a negative regulator of cell proliferation. The gene encoding p57KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome (BWS). Previously we demonstrated that p57KIP2 is imprinted and only the maternal allele is expressed in both mice and humans. We also showed mutations found in p57KIP2 in patients with BWS that were transmitted from the patients' carrier mothers, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. In the study reported here, we performed functional analysis of the two mutated p57KIP2 genes. We showed that the nonsense mutation found in the Cdk inhibitory domain in a BWS patient rendered the protein inactive with consequent complete loss of its role as a cell cycle inhibitor and of its nuclear localization. We also showed that the mutation in the QT domain, although completely retaining its cell cycle regulatory activity, lacked nuclear localization and was thus prevented from performing its role as an active cell cycle inhibitor. Consequently, no active p57KIP2 would have existed, which might have caused the disorders in BWS patients.

Cyclins and cell cycle checkpoints.

The eucaryotic cell cycle is regulated by the periodic synthesis and destruction of cyclins that associate with and activate cyclin-dependent kinases. Cyclin-dependent kinase inhibitors, such as p21 and p16, also play important roles in cell cycle control by coordinating internal and external signals and impeding proliferation at several key checkpoints. Understanding how these proteins interact to regulate the cell cycle has become increasingly important to researchers and clinicians with the discovery that many of the genes that encode cell cycle regulatory activities are targets for alterations that underlie the development of cancer. Several therapeutic agents, such as DNA-damaging drugs, microtubule inhibitors, antimetabolites, and topoisomerase inhibitors, take advantage of this disruption in normal cell cycle regulation to target checkpoint controls and ultimately induce growth arrest or apoptosis of neoplastic cells. Other therapeutic drugs being developed, such as UCN-01, specifically inhibit cell cycle regulatory proteins.

DNA damage-associated cell cycle and cell death control is differentially modulated by caffeine in clones with p53 mutations.

Caffeine is known to potentiate the cytotoxic effects of DNA damaging agents and increases the sensitivity of p53-deficient cells to X-irradiation (X-IR). We have analyzed the cell cycle and cell death control after X-IR in the absence or presence of caffeine in hematological cell lines with various configurations of the p53 gene; EBV-immortalized lymphoblastoid cells with heterozygous p53 mutation (wt/mt), human leukemia cell lines HL60 and KOPM28 with no and mutant p53 expression, respectively. These cell lines display an impaired G0/G1 checkpoint and G2 delay following X-IR, and resistance to apoptosis, which are in accordance with findings previously reported. When irradiated in combination with caffeine, all these cell lines overrode the G2 delay and accumulated at G0/G1. The cell cycle modifications in these cell lines correlated with the increase in radiation-induced p34Cdc2 kinase activity by caffeine. These cell cycle control modifications by caffeine, however, were not associated with enhancement of radiation-induced apoptosis or reduction of clonogenic growth activity in these cell lines. These results suggest that the cytocidal effect of caffeine may need to be verified independently of its cell cycle regulatory activities at least in some cases with p53 mutation.

Control of Apoptosis by Human Adenovirus Genes

Adenovirus infection induces apoptosis by multiple paradigms that involve viral proteins coded by three different early gene regions,E1A, E3,andE4.These cell death programs are antagonized by a different set of viral proteins coded by early gene blocksE1B, E3,andE4.The E1A proteins activate the cellular transcription factor E2F and also increase accumulation of the p53 tumor suppressor protein, both proteins with known apoptotic activity. The E1B-19K protein, a distantly related member of the BCL-2 family of antiapoptosis proteins, efficiently suppresses both p53-dependent and -independent apoptosis induced during adenovirus infection. Two other proteins, E1B-55K and E4-36K, which inactivate p53 by physical protein complex formation suppress p53-dependent apoptosis. The mechanisms by which the E1B-19K protein acts are not fully known, but at least one appears to involve antagonizing the activity of various cellular pro-apoptotic proteins such as BAX, BAK, and BIK. One of the two major E1A proteins, 289R, also appears to induce cell death via transactivation of the cellular transcription factor NF-κB as well as two early gene blocks,E3andE4.NF-κB is believed to induce expression of TNF and various inflammatory cytokines in virus-infected cells. TheE3andE4gene blocks each code for a cell death protein, E3-11.6K (ADP) and E4-orf4. The E1A proteins also sensitize infected cells for TNF-induced apoptosis and this activity is linked to the cell cycle regulatory activities of E1A. In addition to the E1B-19K protein, three E3 proteins, 14.7K, 10.4K, and 14.5K, specifically protect cells against TNF-induced cell death. One of the mechanisms by which the E3-14.7K and E3-10.4K/14.5K complex suppress TNF-induced toxicity appears to be mediated by the release of arachidonic acid by the cytosolic phospholipase A2. The E3 proteins also inhibit Fas-agonist-induced apoptosis by endosome-mediated internalization and degradation of Fas from the cell surface. Suppression of E1A-induced apoptosis in nonpermissive cells by E1B-19K, E1B-55K, and E4-36K proteins leads to oncogenic transformation.

Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin.

The nucleolar protein gar2 of fission yeast is structurally related to the multifunctional nucleolar protein nucleolin from vertebrates and has been shown to be implicated in production of 18S rRNA. gar2 contains several potential casein kinase 2 (CK2) phosphorylation sites and a single putative p34(cdc2 )phosphorylation site in the consensus S50PKK. Here, we show that, like nucleolin, gar2 is phosphorylated in vitro by both highly purified CK2 from CHO cells and p34(cdc2 )from starfish oocytes. Moreover, the substitution of alanine for the N-terminal serine 50 abolishes phosphorylation by p34(cdc2 )in vitro. We also provide evidence that gar2 is phosphorylated in vitro by a p13(suc1)-Sepharose-bound kinase from Schizosaccharomyces pombe extracts that displays cell cycle-regulated activity similar to that of the p34(cdc2(kinase. In vivo 32P labeling of cells indicates that gar2 is a phosphoprotein and that incorporation of phosphate on residue 50 occurs specifically at mitosis. Taken together, these results lead us to propose that gar2 is likely to be an in vivo substrate for the mitotic p34(cdc2 )kinase. However, this posttranslational modification of the gar2 protein does not appear to be essential for normal production of 18S rRNA.