P27 Kip1 Protein Research Articles

Genetic Evidence That Intestinal Notch Functions Vary Regionally and Operate through a Common Mechanism of Math1 Repression

Notch signaling is active in many sites, and its diverse activities must require tissue-specific intermediaries, which are largely unknown. In the intestinal epithelium, Notch promotes crypt cell proliferation and inhibits goblet cell differentiation. Pharmacologic studies suggest that the latter effect occurs through the transcription factor Math1/Atoh1, which specifies all intestinal secretory cells. We tested this hypothesis using mouse mutants. Genetic loss of the Notch effector RBP-Jκ alone increases all intestinal secretory lineages, with variation between proximal and distal gut segments. This secretory cell excess observed with RBP-Jκ loss was blocked in the absence of Math1 in RBP-Jκ(Fl/Fl);Math1(Fl/Fl);Villin-Cre((ER-T2)) mice. Loss of both factors also restored progenitor replication, proving that Math1 is epistatic to Notch signaling in both secretory cell differentiation and crypt cell proliferation. Investigating mechanisms downstream of Math1, we found that expression of the known Notch effector protein Hes1 was predictably lost in RBP-Jκ(-/-) mice but surprisingly recovered in RBP-Jκ;Math1 compound conditional mutants. Furthermore, the cell cycle inhibitors p27(Kip1) and p57(Kip2) were selectively overexpressed in duodenal and ileal crypts, respectively, in RBP-Jκ-deficient mice. Regional activation of these products was completely abrogated in the absence of Math1. Thus, all intestinal Notch effects channel through the tissue-restricted factor Math1, which promotes secretory differentiation and cell cycle exit by regionally distinct mechanisms. Our data further suggest that, besides transmitting Notch signals, the transcription factor Hes1 acts downstream of Math1 to regulate expression of cell cycle inhibitors and intestinal crypt cell replication.

H 2O 2 scavenging inhibits G1/S transition by increasing nuclear levels of p27 KIP1

The aim of the present study was to evaluate cell cycle regulation by scavenging H 2O 2 in tumor cells. A significant arrest in the G1 phase of the cell cycle was demonstrated in CH72-T4 carcinoma cells exposed to catalase, associated with a decrease in cyclin D1 and an increase in the CDK inhibitory protein p27 KIP1. Moreover, we found a differential intracellular distribution of p27 KIP1, which remained in the nucleus after catalase treatment. In vivo experiments showed an increase in nuclear levels of p27 KIP1 associated with the inhibition of tumor growth by H 2O 2 scavenging, confirming in vitro results. To conclude, H 2O 2 scavenging may induce cell cycle arrest through the modulation of cyclin D1 and p27 KIP1 levels and nuclear localization of p27 KIP1. To our knowledge, this is the first report that demonstrates that the modulation of ROS alters the intracellular localization of a key regulatory protein of G1/S transition.

The Atr Protein Kinase Controls UV-Dependent Upregulation of p16INK4A Through Inhibition of Skp2-Related Polyubiquitination/Degradation

The tumor suppressor p16(INK4A), a phosphoprotein that exists in human cells under both phosphorylated and nonphosphorylated forms, plays crucial roles during the cellular response to UV light. However, it is still unclear how this protein is activated in response to this carcinogenic agent. We have shown here that UVC upregulates p16(INK4A) and the phosphorylated form of the protein at the 4 serine sites; Ser-7, Ser-8, Ser-140, and Ser-152. This accumulation of p16(INK4A) occurred through increasing the stability of both forms of the protein. Importantly, phospho-p16(INK4A) showed much higher stability, and UV treatment strongly increased its level in absence of de novo protein synthesis. Furthermore, we have shown that the UV-dependent upregulation of both forms of p16(INK4A) is under the control of the protein kinase Atr, which suppresses their UVC-dependent proteasomal degradation. Interestingly, although this degradation is ubiquitin-related for p16(INK4A) through the Skp2 ubiquitin ligase protein, it is ubiquitin-independent for the phosphorylated form. In addition, we present clear evidence that Skp2 is upregulated in ATR-deficient cells, leading to the downregulation of the p27(Kip1) protein in response to UVC light. Moreover, we have shown a preferential association of endogeneous phospho-p16(INK4A) with Cdk4. This association increased following UV-treatment mainly for p16(INK4A) phosphorylated at Ser-140 and Ser-152. Besides, we have shown that Atr regulates UV-related p16/Cdk4-dependent and -independent phosphorylation of pRB and G1 cell cycle delay. Together, these results indicate that p16(INK4A) and p27(Kip1) are key targets in the Atr-dependent signaling pathway in response to UV damage.

Role of T198 Modification in the Regulation of p27Kip1 Protein Stability and Function

The tumor suppressor gene p27Kip1 plays a fundamental role in human cancer progression. Its expression and/or functions are altered in almost all the different tumor histotype analyzed so far. Recently, it has been demonstrated that the tumor suppression function of p27 resides not only in the ability to inhibit Cyclins/CDKs complexes through its N-terminal domain but also in the capacity to modulate cell motility through its C-terminal portion. Particular interest has been raised by the last amino-acid, (Threonine 198) in the regulation of both protein stability and cell motility.Here, we describe that the presence of Threonine in position 198 is of primary importance for the regulation of the protein stability and for the control of cell motility. However, while the control of cell motility is dependent on the phosphorylation of T198, the stability of the protein is specifically controlled by the steric hindrance of the last amino acid. The effects of T198 modification on protein stability are not linked to the capacity of p27 to bind Cyclins/CDKs complexes and/or the F-box protein Skp2. Conversely, our results support the hypothesis that conformational changes in the disordered structure of the C-terminal portion of p27 are important in its ability to be degraded via a proteasome-dependent mechanism. On the other hand T198 phosphorylation favors p27/stathmin interaction eventually contributing to the regulation of cell motility, supporting the hypothesis that the presence of T198 is fundamental for the regulation of p27 functions.

Targeting p27Kip1 protein: its relevance in the therapy of human cancer

Introduction: Cell division cycle progression is achieved by a sequential and stringently concerted activation of a family of serine–threonine kinases, namely the cyclin-dependent kinases (CDKs). p27Kip1 is a pivotal CDK inhibitor and a tight modulator of CDK-dependent phenotypes. Thus, p27Kip1 plays a fundamental role in key cellular processes such as proliferation, differentiation, apoptosis, substrate adhesion and motility. Intriguingly, when p27Kip1 is localized in the nucleus, it acts as an antiproliferative protein, while, in the cytosol, p27Kip1 promotes cytoskeleton remodeling and might positively influence metastatization. Downregulation of p27Kip1 nuclear level or its cytosolic mislocalization are consistently correlated with poor prognosis of numerous types of human epithelial and non-epithelial cancers.Areas covered: This review illustrates the basic structural features of p27Kip1 protein, its metabolism and alterations in human malignancies, along with describing anticancer strategies based on targeting p27Kip1.Expert opinion: Given the role of p27Kip1 in the control of cell proliferation and its decreased level observed in malignancies with poor outcome, drugs able to handle the protein levels and localization might represent an important goal for novel specific and effective anticancer strategies. Although no convincing proofs have been reported, putative negative consequences of p27Kip1 targeting might be also conceivable.

Prolonged Mechanical Ventilation Induces Cell Cycle Arrest in Newborn Rat Lung

RationaleThe molecular mechanism(s) by which mechanical ventilation disrupts alveolar development, a hallmark of bronchopulmonary dysplasia, is unknown.ObjectiveTo determine the effect of 24 h of mechanical ventilation on lung cell cycle regulators, cell proliferation and alveolar formation in newborn rats.MethodsSeven-day old rats were ventilated with room air for 8, 12 and 24 h using relatively moderate tidal volumes (8.5 mL.kg−1).Measurement and Main ResultsVentilation for 24 h (h) decreased the number of elastin-positive secondary crests and increased the mean linear intercept, indicating arrest of alveolar development. Proliferation (assessed by BrdU incorporation) was halved after 12 h of ventilation and completely arrested after 24 h. Cyclin D1 and E1 mRNA and protein levels were decreased after 8–24 h of ventilation, while that of p27Kip1 was significantly increased. Mechanical ventilation for 24 h also increased levels of p57Kip2, decreased that of p16INK4a, while the levels of p21Waf/Cip1 and p15INK4b were unchanged. Increased p27Kip1 expression coincided with reduced phosphorylation of p27Kip1 at Thr157, Thr187 and Thr198 (p<0.05), thereby promoting its nuclear localization. Similar -but more rapid- changes in cell cycle regulators were noted when 7-day rats were ventilated with high tidal volume (40 mL.kg−1) and when fetal lung epithelial cells were subjected to a continuous (17% elongation) cyclic stretch.ConclusionThis is the first demonstration that prolonged (24 h) of mechanical ventilation causes cell cycle arrest in newborn rat lungs; the arrest occurs in G1 and is caused by increased expression and nuclear localization of Cdk inhibitor proteins (p27Kip1, p57Kip2) from the Kip family.

Somatic Mutation and Germline Sequence Abnormalities inCDKN1B, Encoding p27Kip1, in Sporadic Parathyroid Adenomas

Typical nonfamilial (sporadic) parathyroid adenomas are common endocrine tumors for which no predisposing germline DNA variants and only a few clonally altered genes that drive parathyroid tumorigenesis have been identified. CDKN1B, encoding cyclin-dependent kinase inhibitor p27(kip1), has recently been implicated in a multiple endocrine tumor phenotype in rats and, rarely, in a human familial MEN1 (multiple endocrine neoplasia type 1)-like disorder. We sought to determine whether mutation of CDKN1B might contribute to the development of common sporadic parathyroid adenomas. We sequenced the CDKN1B gene in 86 parathyroid adenomas from patients with typical, sporadic presentations of primary hyperparathyroidism. Identified alterations were categorized as somatic or germline, and their functional consequences were examined. CDKN1B sequence abnormalities were identified in four parathyroid adenomas. Acquired biallelic alteration of CDKN1B, resulting from somatic mutation plus loss of heterozygosity, was detected in one tumor. Germline origin was documented in two cases despite nonfamilial presentations. None of the observed alterations were found in 240 CDKN1B alleles from normal individuals, nor among more than 2,000 previously reported alleles. Most identified variants reduced p27(kip1) protein levels or altered in vitro stability. In typical, sporadic parathyroid adenomas, CDKN1B mutation can be somatic and clonal, indicative of a directly conferred selective advantage in parathyroid tumorigenesis. Additionally, the identification of germline CDKN1B variants in patients with sporadic presentations provides evidence for CDKN1B as a susceptibility gene in the development of typical parathyroid adenomas.

Up-regulation of p27kip1 contributes to Nrf2-mediated protection against angiotensin II-induced cardiac hypertrophy

Nuclear factor erythroid-2-related factor 2 (Nrf2) appears to be a negative regulator of maladaptive cardiac remodelling and dysfunction; however, a potential of the Nrf2-mediated cardiac protection in diverse pathological settings remains to be determined. This study was aimed to explore the role of Nrf2 in angiotensin II (Ang II)-induced cardiac hypertrophy. Littermate wild-type (WT) and Nrf2 knockout (Nrf2(-/-)) mice were administered Ang II via osmotic mini-pumps for 2 weeks to induce cardiac hypertrophy. Elevation of blood pressure by the continuous Ang II infusion was comparable between WT and Nrf2(-/-) mice. Relative to WT mice, however, Nrf2(-/-) mice exhibited exaggerated myocardial oxidative stress with an impaired induction of a group of antioxidant genes and increased cardiac hypertrophy in response to the sustained Ang II stimulation. In cultured cardiomyocytes, adenoviral overexpression of Nrf2 shRNA enhanced Ang II-induced reactive oxygen species (ROS) production and protein synthesis, whereas adenoviral overexpression of Nrf2 exerted opposite effects. Moreover, Nrf2 deficiency exacerbated Ang II-induced down-regulation of p27(kip1) expression in the heart via a mechanism of post-transcriptional regulation. In contrast, adenoviral overexpression of Nrf2 increased p27(kip1) protein but not mRNA expression and reversed Ang II-induced down-regulation of p27(kip1) protein expression in cultured cardiomyocytes by suppressing ROS formation. Finally, the enhancement of Ang II-induced hypertrophic growth due to the Nrf2 deficiency was negated by overexpressing p27(kip1) in cultured cardiomyocytes. The Nrf2-p27(kip1) pathway serves as a novel negative feedback mechanism in Ang II-induced pathogenesis of cardiac hypertrophy, independent of changes in blood pressure.

Damage-specific DNA binding protein 1 (DDB1) is involved in ubiquitin-mediated proteolysis of p27Kip1 in response to UV irradiation

Damage-specific DNA binding protein 1 (DDB1) is a conserved protein component of the damaged DNA binding protein complex (DDB) that recognizes UV-induced DNA lesions and initiates the nucleotide excision repair process. DDB1 is also part of an E3 ubiquitin-ligase complex that targets a variety of substrates for proteolysis including the cyclin-dependent kinase inhibitor p27Kip1. The mechanism regulating the trafficking of DDB1 and its relationship with UV irradiation is not known, although cell cycle progression is implicated in the molecular machinery driving DDB1 into the nucleus. We evaluated the involvement of DDB1 in ubiquitination of the cdk inhibitor p27Kip1 in response to UV irradiation. First, we observed that low and high doses of UV irradiation exert different effects on p27Kip1 protein levels. Indeed, low but not high UV doses induced p27Kip1 protein proteolysis in several human cell lines and UV-dependent degradation is dominant over other genotoxic agents such as cisplatin. We also demonstrate that p27Kip1 reduction is not due to transcriptional regulation and that the proteasome inhibitor MG132 affects p27Kip1 degradation.We observed that at low UV doses the decrease in p27Kip1 nuclear protein related with DDB1 translocation into the nucleus; conversely, high doses of UV-induced p27Kip1 accumulation and unchanged level of DDB1. The knockdown of DDB1 or Skp2 prevents UV-induced degradation of p27Kip1 suggesting that DDB1 is essential to regulation of p27kip1 turnover after a mild DNA damage. Our findings support the concept that DDB1 contributes to the activation of DNA repair mechanisms and could be a key factor in regulating the cell cycle in response to UV-induced DNA damage. Although the temporal order with which DDB1 contributes to ubiquitination of p27Kip1 or initiates the nucleotide excision repair process remains to be established, our results represent a major step towards clarifying these issues.

Upstream molecular signaling pathways of p27(Kip1) expression in human breast cancer cells in vitro: differential effects of 4-hydroxytamoxifen and deficiency of either D-(+)-glucose or L-leucine.

BackgroundThe objective of this study was to investigate whether the levels of glucose or certain amino acids could regulate the expression of a cell cycle repressor protein p27(Kip1), thereby dictating the risk of cancer in either obesity or caloric/dietary restriction. Previously, we identified and reported four different upstream molecular signaling pathways of p27 expression in human breast cancer cells. We called these four pathways as pathway #1, #2, #3 and #4. We found that 4-hydroxytamoxifen - but not tamoxifen - up-regulated the expression of p27 using pathway #1 which consisted mainly of receptor tyrosine kinases and mTORC1. We now investigate, using 4-hydroxytamoxifen as a reference anti-cancer agents, whether (a) the moderate increase in the concentration of D-(+)-glucose could down-regulate and, conversely, (b) the deficiency of D-(+)-glucose or certain L-amino acids could up-regulate the expression of p27 in these cells using pathway #2 which consists mainly of AMPK and mTORC1.ResultsUsing human MDA-MB-231 breast cancer cells in vitro, these hypotheses were tested experimentally by performing p27-luciferase reporter transfection assays and western immunoblot analyses. The results obtained are consistent with these hypotheses. Furthermore, the results indicated that, although 4-hydroxytamoxifen used primarily pathway #1 to down-regulate the phosphorylation of 4E-BP1 and up-regulate the expression of p27, it also secondarily down-regulated the phosphorylation of S6K1. In contrast, the deficiency of D-(+)-glucose or L-leucine used primarily pathway #2 to down-regulate the phosphorylation of S6K1, but they also secondarily down-regulated the phosphorylation of 4E-BP1 and up-regulated the expression of p27. Finally, deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A and SIRT3.Conclusions(a) 4-Hydroxitamoxifen used primarily pathway #1 to up-regulate the expression of p27. (b) Moderate increase in the concentration of D-(+)-glucose used primarily pathway #2 to down-regulate the expression of p27. (c) Deficiency of D-(+)-glucose or L-leucine also used primarily pathway #2 to up-regulate the expression of p27. (d) Deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A in the Complex V of respiratory oxidation-phosphorylation chain and mitochondrial SIRT3. The SIRT3 is one of the seven mammalian anti-aging as well as anti-metabolic sirtuins.

Effects of an Indolocarbazole-Derived CDK4 Inhibitor on Breast Cancer Cells

Cyclin D1 (D1) binds to cyclin-dependent kinases (CDK) 4 or 6 to form a holoenzyme that phosphorylates the Rb protein to promote cell cycle progression from G1 to S phase. Therefore, targeting CDK4/6 may be a good strategy for chemotherapy of cancer. We performed a proof-of-principle study to determine the effect of Naphtho [2, 1-α] pyrrolo [3, 4-c] carbazole-5, 7 (6H, 12H)-dione (NPCD), a novel CDK4 inhibitor, on breast cancer cell lines. NPCD was synthesized and purified to over 99% purity verified by HPLC. MCF7, MB231, MCF15, T47D and GI101Ap human breast cancer cells were analyzed for the efficacy of NPCD with MTT and clonogenic assays, with FACS and staining for ethidium bromide and acridine orange for cell death and cell cycle profile. Western blot, reverse transcription and PCR were used for studies of gene expression, and co-immunoprecipitation for protein-complex formation. MTT assay showed that NPCD caused growth arrest and apoptosis of MCF7, MDA-MB231, T47D, MCF15 and GI101Ap cells with an IC50 ranging between 3 to 8 µM given as a single dose. The growth arrest persisted for many days after cessation of the treatment, as shown in a clonogenic assay. NPCD could induce or reduce the D1 and CDK4 protein levels, depending on the cell line, but this effect was not correlated with its efficacy. Phosphorylation of D1 at Thr286 was decreased but it unexpectedly did not correlate with the change in D1 level in the cell lines studied. Phosphorylation of the Rb protein was decreased as expected whereas the p27kip1 protein level was decreased unexpectedly. Protein levels of p21cip1, CDK2 and cyclin E were also decreased in some, but not all, of the cell lines, whereas the mRNA levels of D1, CDK4, cyclin E, CDK2, p27kip1 and p21cip1 were increased in different cell lines. NPCD can cause long-lasting growth arrest and cell death of breast cancer cell lines at an IC50 of 3-8 µM. Decreased phosphorylation of Rb by D1-CDK4/6 and decreased p27kip1 protein level may be part of the underlying mechanism.

Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background

Human germ cell tumors show a strong sensitivity to genetic background similar to Dnd1Ter/Ter mutant mice, where testicular teratomas arise only on the 129/SvJ genetic background. The introduction of the Bax mutation onto mixed background Dnd1Ter/Ter mutants, where teratomas do not typically develop, resulted in a high incidence of teratomas. However, when Dnd1Ter/Ter; Bax–/– double mutants were backcrossed to C57BL/6J, no tumors arose. Dnd1Ter/Ter germ cells show a strong downregulation of male differentiation genes including Nanos2. In susceptible strains, where teratomas initiate around E15.5-E17.5, many mutant germ cells fail to enter mitotic arrest in G0 and do not downregulate the pluripotency markers NANOG, SOX2 and OCT4. We show that DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including p27Kip1 and p21Cip1. P27Kip1 and P21Cip1 protein are both significantly decreased in Dnd1Ter/Ter germ cells on all strain backgrounds tested, strongly suggesting that DND1 regulates mitotic arrest in male germ cells through translational regulation of cell cycle genes. Nonetheless, in C57BL/6J mutants, germ cells arrest prior to M-phase of the cell cycle and downregulate NANOG, SOX2 and OCT4. Consistent with their ability to rescue cell cycle arrest, C57BL/6J germ cells overexpress negative regulators of the cell cycle relative to 129/SvJ. This work suggests that reprogramming of pluripotency in germ cells and prevention of tumor formation requires cell cycle arrest, and that differences in the balance of cell cycle regulators between 129/SvJ and C57BL/6 might underlie differences in tumor susceptibility.

P27Kip1 Negatively Regulates the Activation of Murine Primordial Oocytes

In mice, small oocytes (primordial oocytes) are enclosed within flattened granulosa cells to form primordial follicles around birth. A small number of primordial oocytes enter the growth phase, whereas others are quiescent. The mechanism regulating this selection of primordial oocytes is not well understood. The objective of the present study was to understand the role of p27(Kip1), which regulates cell cycle progression in somatic cells, in the growth initiation of primordial oocytes in neonatal mice. We studied the localization of p27(Kip1) in 0-, 3-, 5-, 7- and 21-day-old mouse ovaries by immunohistochemistry. Ovaries from 3-day-old mice were treated with p27(Kip1) siRNAs (small interfering RNAs), and knockdown of p27(Kip1) was determined by immunohistochemistry and Western blotting. Ovaries treated with siRNAs were organ-cultured for 6 days, and oocyte growth was estimated histologically. Expression of p27(Kip1) was undetectable in the primordial oocytes of newborn mice. In the 3-day-old ovaries (n=3), p27(Kip1) was demonstrated in the nucleus of 36 ± 6% primordial oocytes. The percentage of p27(Kip1)-positive primordial oocytes increased to 72 ± 8 (n=3), 85 ± 7 (n=3) and 93 ± 5 (n=3) in the 5-, 7- and 21-day-old mouse ovaries, respectively. After knockdown of the p27(Kip1) protein by siRNAs, a higher proportion of oocytes entered the growth phase in cultured ovaries than those in the control. These results suggest that p27(Kip1) negatively regulates primordial oocyte growth and that knockdown of p27(Kip1) leads primordial oocytes to enter the growth phase in vitro.

Expression of p27kip1 and skp2 protein in lung carcinoma tissue

Objective To explore the relationship between the expression of S-phase kinase-associated protein 2 ( skp2),cyclin dependent kinase inhibitor protein 27 ( p27kip1) and lung carcinogenesis.Methods The protein expression of p27kip1 and skp2 was detected by immunohistochemistry ( SP method)in 56 cases of lung carcinoma and 19 cases of paraneoplastic normal tissues as control group.Results The positive expression rate of p27kip1 protein was 46.42% in lung carcinoma,while that was 89.47% in paraneoplastic normal tissue ( P ＜0.01 ).The positive expression rate of skp2 protein was 32.14% in lung carcinoma,while that was 10.52% in paraneoplastic normal tissue (P ＜ 0.05 ).There was a negative correlation between the expression of p27kip1 and skp2 in lung corcinoma ( P ＜ 0.01 ).Conclusion The detection of p27kip1 and skp2 proteins is helpful to clinical diagnosis and prognostic evaluation of lung corcinoma. Key words: Lung carcinoma; Cyclin; skp2

Arsenic trioxide-mediated growth inhibition of myeloma cells is associated with an extrinsic or intrinsic signaling pathway through activation of TRAIL or TRAIL receptor 2

Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation. Preclinical and clinical studies showed that ATO has anti-myeloma effects. However, the underlying mechanism remains elusive. In this study, the molecular mechanisms of ATO-induced myeloma apoptosis were explored on four myeloma cell lines of wild type or mutant p53 status and also on six primary myeloma cells. ATO induced potent inhibition of myeloma cell growth and myeloma cell apoptosis compared with controls. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb while up-regulating p53, p21Cip1, and p27Kip1 proteins, resulting in G0/G1 or G2/M cell cycle arrest. ATO treatment increased mRNA levels of interferon regulatory factor-1 and TRAIL, as well as protein levels of caspase 8 and cleaved caspase 3, indicating the involvement of the extrinsic apoptotic pathway in the mutated p53 myeloma cells. ATO also activated caspases 3 and 9, indicating involvement of the intrinsic apoptotic pathway in the wild type p53 myeloma cells. More importantly, these molecular changes induced by ATO-treated myeloma cells are very similar to the baseline expression pattern of hyperdiploid myeloma, which has a relative good prognosis with high expression of TRAIL and interferon related genes. Together, our data suggest that ATO induces apoptosis in MM through either extrinsic or intrinsic signaling pathway, depending on the p53 genetic background. These observations may be employed as prognostic tools and lead to novel therapies in primary myelomas.

TRAIL as the ATO-Target Gene Uniquely Activated In the Hyperdiploid Subtype of Myeloma with Prognostic Relevance, Resulting In Better Prognosis

AbstractAbstract 1904Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying mechanism remains elusive. In this study, the molecular mechanisms of ATO-induced myeloma apoptosis were explored on four myeloma cell lines OPM2, U266, RPMI8226, and KMS28PE of wild type or mutant p53 status and six primary myeloma cells. Gene expression profiling (GEP) of CD138+ bone marrow plasma cells from 22 healthy individuals (NPC), 44 patients with monoclonal gammopathy of undetermined significance (MGUS), and 351 newly diagnosed MM patients were published previously (Zhan et al. Blood. 2006;108:2020-8. Shaughnessy et al. Blood. 2007;109:2276-84.); and GEP from 9 myeloma cell lines were used in this study from the unpublished data of the University of Utah. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. GEP was performed by using the Affymetrix U133Plus2.0 microarray. ATO induced potent inhibition of myeloma cell growth and myeloma cell apoptosis, compared with controls. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb while up-regulating p53, p21Cip1, and p27Kip1 proteins, resulting in G0/G1 or G2/M cell cycle arrest. ATO treatment increased mRNA levels of interferon regulatory factor-1 and TRAIL, as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway in the mutated p53 myeloma cells. ATO also activated caspases 3 and 9, indicating involvement of the intrinsic apoptotic pathway in the wild type p53 myeloma cells. The usage of ATO and TRAIL agonist together has a synergistic effect, indicated by a combination index of less than 1. More importantly, these molecular changes induced by ATO-treated myeloma cells are very similar to the baseline expression pattern of hyperdiploid myeloma, which has a relative good prognosis with high expression of TRAIL and interferon related genes. Together, our data suggest that ATO induces apoptosis in MM through either extrinsic or intrinsic signaling pathway depending on the p53 genetic background. These observations may be employed as prognostic tools and lead to novel therapies in primary myelomas. AcknowledgmentsThis work was supported by grants from National Natural Science Foundation of China (30973450 to JS), start-up funds from Shanghai Tenth People's Hospital (JS), institutional start-up funds from the University of Utah School of Medicine and the Huntsman Cancer Institute (FZ), the National Institutes of Health grant RO1 (CA115399 to GT, FZ) and Senior Award from the Multiple Myeloma Research Foundation (FZ). Disclosures:Zhan:University of Utah: Employment, patent Submission.

Ceramide Produces Apoptosis Through Induction of p27kip1 by Protein Phosphatase 2A-dependent Akt Dephosphorylation in PC-3 Prostate Cancer Cells

Ceramide induces cell cycle arrest and apoptotic cell death associated with increased levels of p27kip1. The aim of this study was to examine the effects of ceramide on p27kip1 protein levels as a measure of cell cycle arrest and apoptosis. Results showed that ceramide increased p27kip1 protein levels through activation of protein phosphatase 2A (PP2A) in PC-3 prostate cancer cells. Treatment of cells with the PP2A inhibitor okadaic acid or with PP2A-Cα siRNA inhibited ceramide-induced enhanced p27kip1 protein expression and Akt dephosphorylation, and prevented Skp2 downregulation. Overexpression of constitutively active Akt attenuated ceramide-induced Skp2 downregulation and p27kip1 upregulation. In addition, ceramide stimulated binding of the PP2A catalytic subunit PP2A-Cαβ to Akt as assessed by immunoprecipitation experiments, indicating that PP2A is involved in the induction of p27kip1 via inhibition of Akt pathway. Finally, whether PP2A can regulate p27kip1 expression independently of Akt pathway was determined. Knockdown of PP2A-Cα with siRNA reduced p27kip1 levels in the presence of Akt inhibitor. These data reveal that PP2A is a regulator of ceramide-induced p27kip1 expression via Akt-dependent and Akt-independent pathways.

Leg Amputation Accelerates Senescence of Rat Lumbar Intervertebral Discs

Several senescence biomarkers were observed to investigate cell senescence in degenerative intervertebral lumbar discs of foreleg-amputated rats. To determine if cell senescence is accelerated in degenerative intervertebral lumbar disc cells in an upright-rat model. Cellular senescence was accelerated in human and sand rat degenerative intervertebral disc (IVD) cells. Repeated use of upright posture by rats contributed to degenerative disc changes. No convincing evidence of cell senescence was observed in the lumbar disc of the foreleg amputated rat. The forelimbs of 20 rats were amputated at 1 month of age such that they maintained an upright stance; rats were housed in custom-made cages. Nonamputated rats, also 1 month of age, were kept in regular cages and served as a control group. The lumbar IVDs were harvested from rats in 2 groups, at 5 or 9 months following amputation. Senescence-associated-β-galactosidase-positive staining was used to detect cell senescence. p16INK4a and p27KIP were assessed by immunohistochemistry analysis. Total RNA isolated from these samples was used to measure the gene expression of p16INK4a, RB, cyclin D1, CDK4, PTEN, p27KIP, p19ARF, p21, TERT, and RAGE by real-time polymerase chain reaction assay. The highest levels of SA-β-GAL activity were detected in 9-month amputated rats. Quantitative immunohistochemical analysis showed that there were highest rates of p16INK4a and p27KIP protein expression in the cartilage endplate and anulus fibrosus of 9-month amputated rats. The mRNA levels of p16INK4a, RB, PTEN, p27KIP, p19ARF, and RAGE were upregulated. The increased cyclin D1 mRNA level was statistically significant only at the ninth month following amputation; CDK4 and TERT mRNA levels were downregulated to a similar extent at both points compared with nonamputated controls. mRNA expression of p21 was significantly downregulated. Accelerated cell senescence was associated with forelimb amputation that causes abnormal loading in rat lumbar IVDs.

Area-specific dynamics of proliferation and migration during primate corticogenesis: Role of the modular protein p27kip1

Area-specific dynamics of proliferation and migration during primate corticogenesis: Role of the modular protein p27kip1

Cytostatic response of HepG2 to 0.57 MHz electric currents mediated by changes in cell cycle control proteins

The capacitive-resistive electric transfer (CRet) therapy is a non-invasive technique that applies electrical currents of 0.4-0.6 MHz to the treatment of musculoskeletal injuries. Although this therapy has proved effective in clinical studies, its interaction mechanisms at the cellular level still are insufficiently investigated. Results from previous studies have shown that the application of CRet currents at subthermal doses causes alterations in cell cycle progression and decreased proliferation in hepatocarcinoma (HepG2) and neuroblastoma (NB69) human cell lines. The aim of the present study was to investigate the antiproliferative response of HepG2 to CRet currents. The results showed that 24-h intermittent treatment with 50 µA/mm(2) current density induced in HepG2 statistically significant changes in expression and activation of cell cycle control proteins p27Kip1 and cyclins D1, A and B1. The chronology of these changes is coherent with that of the alterations reported in the cell cycle of HepG2 when exposed to the same electric treatment. We propose that the antiproliferative effect exerted by the electric stimulus would be primarily mediated by changes in the expression and activation of proteins intervening in cell cycle regulation, which are among the targets of emerging chemical therapies. The capability to arrest the cell cycle through electrically-induced changes in cell cycle control proteins might open new possibilities in the field of oncology.