HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

Background Human homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated. Methods Real time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively. Results Overexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle. Conclusion The present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.

Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint

Upon the incidence of DNA stress, the ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signaling kinases activate a transient cell cycle arrest that allows cells to repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly conserved over species, the mechanisms by which plant cells stop their cell cycle in response to the loss of genome integrity are unclear. We demonstrate that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is transcriptionally activated upon the cessation of DNA replication or DNA damage in an ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA stress signaling, WEE1-deficient plants showed no obvious cell division or endoreduplication phenotype when grown under nonstress conditions but were hypersensitive to agents that impair DNA replication. Induced WEE1 expression inhibited plant growth by arresting dividing cells in the G2-phase of the cell cycle. We conclude that the plant WEE1 gene is not rate-limiting for cycle progression under normal growth conditions but is a critical target of the ATR-ATM signaling cascades that inhibit the cell cycle upon activation of the DNA integrity checkpoints, coupling mitosis to DNA repair in cells that suffer DNA damage.

Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1

Expression of the DMP1 transcription factor, a cyclin D-binding Myb-like protein, induces growth arrest in mouse embryo fibroblast strains but is devoid of antiproliferative activity in primary diploid fibroblasts that lack the ARF tumor suppressor gene. DMP1 binds to a single canonical recognition site in the ARF promoter to activate gene expression, and in turn, p19ARF synthesis causes p53-dependent cell cycle arrest. Unlike genes such as Myc, adenovirus E1A, and E2F-1, which, when overexpressed, activate the ARF-p53 pathway and trigger apoptosis, DMP1, like ARF itself, does not induce programmed cell death. Therefore, apart from its recently recognized role in protecting cells from potentially oncogenic signals, ARF can be induced in response to antiproliferative stimuli that do not obligatorily lead to apoptosis.

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Cell-cycle arrest and apoptosis hypersusceptibility as a consequence of Lck deficiency in nontransformed T lymphocytes

By using antisense RNA, Lck-deficient transfectants of a T helper 2 (Th2) clone have been derived and shown to have a qualitative defect in the T cell receptor signaling pathway. A striking feature observed only in Lck-deficient T cells was the presence of a constitutively tyrosine-phosphorylated 32-kDa protein. In the present study, we provide evidence that this aberrantly hyperphosphorylated protein is p34cdc2 (cdc2) a key regulator of cell-cycle progression. Lck-deficient transfectants expressed high levels of cdc2 protein and its regulatory units, cyclins A and B. The majority of cdc2, however, was tyrosine-phosphorylated and therefore enzymatically inactive. The transfectants were significantly larger than the parental cells and contained 4N DNA. These results establish that a deficiency in Lck leads to a cell-cycle arrest in G2. Moreover, transfected cells were hypersusceptible to apoptosis when activated through the T cell receptor. Importantly, however, this hypersusceptibility was largely reversed in the presence of T cell growth factors. These findings provide evidence that, in mature T lymphocytes, cell-cycle progression through the G2–M check point requires expression of the Src-family protein tyrosine kinase, Lck. This requirement is Lck-specific; it is observed under conditions in which the closely related Fyn kinase is expressed normally, evincing against a redundancy of function between these two kinases.

CENP-E is an essential kinetochore motor in maturing oocytes and is masked during Mos-dependent, cell cycle arrest at metaphase II

CENP-E, a kinesin-like protein that is known to associate with kinetochores during all phases of mitotic chromosome movement, is shown here to be a component of meiotic kinetochores as well. CENP-E is detected at kinetochores during metaphase I in both mice and frogs, and, as in mitosis, is relocalized to the midbody during telophase. CENP-E function is essential for meiosis I because injection of an antibody to CENP-E into mouse oocytes in prophase completely prevented progression of those oocytes past metaphase I. Beyond this, CENP-E is modified or masked during the natural, Mos-dependent, cell cycle arrest that occurs at metaphase II, although it is readily detectable at the kinetochores in metaphase II oocytes derived from mos-deficient (MOS−/−) mice that fail to arrest at metaphase II. This must reflect a masking of some CENP-E epitopes, not the absence of CENP-E, in meiosis II because a different polyclonal antibody raised to the tail of CENP-E detects CENP-E at kinetochores of metaphase II-arrested eggs and because CENP-E reappears in telophase of mouse oocytes activated in the absence of protein synthesis.

c-Abl-induced apoptosis, but not cell cycle arrest, requires mitogen-activated protein kinase kinase 6 activation

c-Abl is a ubiquitously expressed protein tyrosine kinase activated by DNA damage and implicated in two responses: cell cycle arrest and apoptosis. The downstream pathways by which c-Abl induces these responses remain unclear. We examined the effect of overexpression of c-Abl on the activation of mitogen-activated protein kinase pathways and found that overexpression of c-Abl selectively stimulated p38, while having no effect on c-Jun N-terminal kinase or on extracellular signal-regulated kinase. c-Abl-induced p38 activation was primarily mediated by mitogen-activated protein kinase kinase (MKK)6. A C-terminal truncation mutant of c-Abl showed no activity for stimulating p38 and MKK6, while a kinase-deficient c-Abl mutant still retained a residual activity. We tested different forms of c-Abl for their ability to induce apoptosis and found that apoptosis induction correlated with the activation of the MKK6-p38 kinase pathway. Importantly, dominant-negative MKK6, but not dominant-negative MKK3 or p38, blocked c-Abl-induced apoptosis. Because overexpression of p38 blocks cell cycle G1/S transition, we also tested whether the MKK6-p38 pathway is required for c-Abl-induced cell cycle arrest, and we found that neither MKK6 nor p38 dominant-negative mutants could relieve c-Abl-induced cell cycle arrest. Finally, DNA damage-induced MKK6 and p38 activation was diminished in c-Abl null fibroblasts. Our study suggests that c-Abl is required for DNA damage-induced MKK6 and p38 activation, and that activation of MKK6 by c-Abl is required for c-Abl-induced apoptosis but not c-Abl-induced cell cycle arrest.

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

DNA damage and p53-mediated cell cycle arrest: A reevaluation

Most mammalian cells exhibit transient delays in the G1 and G2 phases of the cell cycle after treatment with radiation or radiomimetic compounds. p53 is required for the arrest in G1, which provides time for DNA repair. Recently, a role of p53 in the G2/M transition has also been suggested. However, it has been reported that the presence of functional p53 does not always correlate with the induction of these checkpoints. To precisely assess the role of p53 in activating cell cycle checkpoints and in cell survival after radiation, we studied the response of two isogenic human fibrosarcoma cell lines differing in their p53 status (wild type or mutant). We found that when irradiated cells undergo a wild-type p53-dependent G1 arrest, they do not subsequently arrest in G2. Moreover, wild-type p53 cells irradiated past the G1 checkpoint arrest in G2 but do not delay in the subsequent G1 phase. Furthermore, in these cell lines, which do not undergo radiation-induced apoptosis, the wild-type p53 cell line exhibited a greater radioresistance in terms of clonogenic survival. These results suggest that the two checkpoints may be interrelated, perhaps through a control system that determines, depending on the extent of the damage, whether the cell needs to arrest cell cycle progression at the subsequent checkpoint for further repair. p53 could be a crucial component of this control system.

The catalytic subunit of DNA-dependent protein kinase selectively regulates p53-dependent apoptosis but not cell-cycle arrest

DNA damage induced by ionizing radiation (IR) activates p53, leading to the regulation of downstream pathways that control cell-cycle progression and apoptosis. However, the mechanisms for the IR-induced p53 activation and the differential activation of pathways downstream of p53 are unclear. Here we provide evidence that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) serves as an upstream effector for p53 activation in response to IR, linking DNA damage to apoptosis. DNA-PKcs knockout (DNA-PKcs−/−) mice were exposed to whole-body IR, and the cell-cycle and apoptotic responses were examined in their thymuses. Our data show that IR induction of apoptosis and Bax expression, both mediated via p53, was significantly suppressed in the thymocytes of DNA-PKcs−/− mice. In contrast, IR-induced cell-cycle arrest and p21 expression were normal. Thus, DNA-PKcs deficiency selectively disrupts p53-dependent apoptosis but not cell-cycle arrest. We also confirmed previous findings that p21 induction was attenuated and cell-cycle arrest was defective in the thymoctyes of whole body-irradiated Atm−/− mice, but the apoptotic response was unperturbed. Taken together, our results support a model in which the upstream effectors DNA-PKcs and Atm selectively activate p53 to differentially regulate cell-cycle and apoptotic responses. Whereas Atm selects for cell-cycle arrest but not apoptosis, DNA-PKcs selects for apoptosis but not cell-cycle arrest.

PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27KIP1 and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27KIP1 and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27KIP1.

Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): Potential role of cyclin D1

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G1 phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 μM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G1 phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G1 phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G1 phase of the cell cycle.

p53-dependent cell cycle arrest induced by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts.

Proteases are known to play important roles in cell growth control, although the underlying mechanisms are still poorly understood. Here we show that the protease inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal induced cell cycle arrest in platelet-derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome. Inhibition of the proteasome resulted in accumulation of the tumor suppressor p53, which was followed by an increase in the amount of the cyclin-dependent kinase-inhibitor p21. As a consequence, both phosphorylation and activity of the cyclin-dependent kinase 2/cyclin E complex were inhibited. We further observed that the retinoblastoma gene product, pRb, remained in the hypophosphorylated state, thus preventing cells from progression into the S-phase. These studies strongly support the hypothesis that the proteasome is a key regulator in the G1-phase of cell cycle progression.

A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F.

Brca1 inactivation induces p27Kip1-dependent cell cycle arrest and delayed development in the mouse mammary gland

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.