Altered Cell Cycle Regulation Research Articles

Molecular mechanism underlying the functional loss of cyclindependent kinase inhibitors p16 and p27 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common human cancers, and its incidence is still increasing in many countries. The prognosis of HCC patients remains poor, and identification of useful molecular prognostic markers is required. Many recent studies have shown that functional alterations of cell-cycle regulators can be observed in HCC. Among the various types of cell-cycle regulators, p16 and p27 are frequently inactivated in HCC and are considered to be potent tumor suppressors. p16, a G1-specific cell-cycle inhibitor that prevents the association of cyclindependent kinase (CDK) 4 and CDK6 with cyclin D1, is frequently inactivated in HCC via CpG methylation of its promoter region. p16 may be involved in the early steps of hepatocarcinogenesis, since p16 gene methylation has been detected in subsets of pre-neoplastic liver cirrhosis patients. p27, a negative regulator of the G1-S phase transition through inhibition of the kinase activities of Cdk2/cyclin A and Cdk2/cyclin E complexes, is now considered to be an adverse prognostic factor in HCC. In some cases of HCC with increased cell proliferation, p27 is overexpressed but inactivated by sequestration into cyclin D1-CDK4-containing complexes. Since loss of p16 is closely related to functional inactivation of p27 in HCC, investigating both p16 and p27 may be useful for precise prognostic predictions in individuals with HCC.

CS1, a New Surface Target on Multiple Myeloma (MM) Cells, Protects Myeloma Cells from Apoptosis Via Regulation of ERK1/2, AKT and STAT3 Signaling Cascades.

Cell surface glycoprotein CS1 is universally expressed in the majority of MM patients. CS1 gene is localized on chromosome 1q which amplified in many MM patients and in MM cell lines (i.e., H929 and OPM2). We recently further detected CS1 protein in MM patient sera, but not in individuals with monoclonal gammopathy of undetermined significance (MGUS) or in healthy donors (Abstract # 950059). Since circulating CS1 levels correlate with active MM (Abstract # 950059), we postulated that CS1 may regulate MM cell growth and survival. We here defined a role of CS1 in MM cell growth and survival by generating CS1-null OPM2 MM cells using lentiviral CS1 short interfering RNA (CS1 siRNA). Four CS1-null OPM2 transfectants were derived using 4 CS1 siRNA targeting different regions in CS1 gene. Specific knockdown was confirmed by complete abrogation of CS1 mRNA and protein expression. In contrast, CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cnt-OPM2). Significantly, immunoblotting analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in all 4 CS1-null OPM2 cells, compared with OPM2 and cnt-OPM2 cells. We next examined growth and survival of these cells using MTT and Alamar Blue colorimetric/fluorescence assays. Serum deprivation markedly induced apoptosis at earlier time points in CS1-null OPM2 cells, but not in cnt-OPM2 cells. Earlier apoptosis in CS1-null OPM2 cells was mediated by earlier activation and cleavage of caspases, i.e. caspase 3 (> 7-fold) and caspase 8 (> 10-fold). Microarray gene expression profiling of cells under normal cultures showed altered cell cycle regulators (i.e., decreased CDK1, cyclin B1, cdc25b, cyclin D2, and increased CDK inhibitor p18), reduced survival/anti-apoptotic proteins (i.e., Mcl-1), and increased proapoptotic molecules (i.e., BINP3, BIM) in CS1-null OPM2 vs. cnt-OPM2 cells (>4-fold alteration, p<0.001). Additionally, p-21-activated kinase 1 (PAK1) that regulates cell motility, invasiveness, and survival in breast cancer cells, was completely blocked in CS1-null OPM2 transfectants. These transcriptional changes were further confirmed by immunoblotting using specific Abs. Moreover, CS1-null OPM2 transfectants had defective IL-6/IL-6R signaling, since IL-6-induced pERK1/2, pAKT, and pSTAT3 was completely blocked in CS1-null cells, but not in cnt-OPM2 cells. FGFR3 was overexpressed in cnt-OPM2 cells but was significantly downregulated in CS1-null OPM2 cells. MIP-1alpha (CCL3, an osteoclasts-activating factor), a downstream target of FGFR3 and ERK1/2 signaling, was concomitantly and specifically downregulated in CS1-null OPM2 transfectants. Ingenuity analysis further revealed altered transcriptional factors (i.e., XBP-1 and its downstream target genes, IRF4, RUNX2, SMAD1), immunoproteasome components (i.e., NMI, PSMB9, PSME2), and cell surface receptors (i.e., CCR1, Syndecan 1, IL-6R, gp130) in CS1-null vs cnt-OPM2 cells. Finally, MM1S cells transfected with a CS1-overexpressing plasmid had a moderate growth increase and increased pERK1/2, pAKT, and pSTAT3, compared with parental MM1S cells. Together, these results suggest a role of CS1 in MM cell survival by cross-talking major growth and survival pathways, strongly supporting novel therapies targeting CS1 function in MM.

Expression of MDM2, p16, and HPV Status is Associated with Survival after Radiotherapy in Patients with Carcinoma of the Cervix

The majority of cervix cancers are attributed to effects of HPV viral infection, which may cause cellular dysregulation via viral associated protein interaction with p53 and Rb resulting in p16 over-expression as a cellular compensatory mechanism. It is possible that alternate mechanisms of cervical carcinogenesis exist that are independent of the viral effects. These may include p53 mutation, or alteration of cell cycle regulators, such as over-expression of the p53 inhibitor MDM2.

Expression of the high mobility group A family member p8 is essential to maintaining tumorigenic potential by promoting cell cycle dysregulation in LβT2 cells

The mechanism by which the HMGA protein p8 facilitates tumorigenesis may be cell cycle dysregulation. Control- (C) LβT2 cells, which express p8, form tumors at a rate five-times faster than p8-knockdown (p8-KD)-LβT2 cells. In association with this heightened tumorigenic potential, p8-expressing C-LβT2 cells avoid G 0/G 1 arrest and become genetically unstable while p8-KD-LβT2 cells arrest in G 0/G 1, become senescent upon overgrowth, and maintain a diploid population. These phenotypic changes correspond to altered cell cycle regulation at the G 1-to-S transition that may be due to p8-mediated changes in expression of the Cip/Kip family members of cell cycle inhibitors, p21, p27, and p57.

Molecular Mechanisms in Barrett’s Metaplasia and Its Progression

The dramatic increase in the incidence and poor overall survival rates of esophageal/gastroesophageal junction adenocarcinoma underscore the necessity to discover molecular markers that can be used for risk assessment, early diagnosis, and targeted therapeutic intervention. Barrett's esophagus (BE) is proposed to represent a precursor of esophageal/gastroesophageal junction adenocarcinoma. BE progression to invasive cancer is defined by a metaplasia-dysplasia-carcinoma progression characterized by an increasing accumulation of genetic changes associated with alterations in molecular gatekeepers of cell circuitries and tissue homeostasis. Using a combination of in situ tissue-based and high-throughput analyses, we investigated alterations of cell-cycle regulators and inflammation-associated molecular effectors. Our data suggest a potential synergistic effect of these alterations for the BE progression to cancer, and underscore the potential use of these markers: (1) in molecular panels assessing cancer risk in BE patients; and (2) as potential therapeutic targets for chemopreventive interventions and to enhance response to anti-neoplastic therapies.

Elevated risk of squamous-cell carcinoma of the lung in heavy smokers carrying the variant alleles of the TP53 Arg72Pro and p21 Ser31Arg polymorphisms

Alterations in cell cycle regulation and apoptosis leading to malignant transformation could be caused by common genetic variants in tumor suppressor genes. The effects of the TP53 polymorphism Arg72Pro on lung cancer risk have been investigated in numerous studies with, however, conflicting results. In many studies, important risk modifiers such as smoking or tumor histology were not taken into account. We therefore investigated the combined effects of polymorphisms in TP53 (Arg72Pro) and p21/CDKN1A (Ser31Arg) and smoking on lung cancer risk. Our case-control study consisted of 405 patients with lung cancer, mainly squamous-cell carcinoma (185) and adenocarcinoma (177) and 404 unmatched tumor-free hospital controls. Multivariate regression analysis showed a moderate but statistically significant risk of lung cancer overall and especially of squamous-cell carcinoma (OR, 1.65; CI, 1.10-2.47) for TP53 72Pro allele carriers. The risk was markedly increased in heavy smokers (>20 pack-years) with squamous-cell carcinoma (OR, 2.80 in patients homozygous for 72Pro; CI, 1.19-6.58), but not in light smokers (<or=20 pack-years). The results for the p21 Ser31Arg polymorphism suggested that 31Ser is a moderate-risk allele for squamous-cell carcinoma. Analysis of the combined effects of the two polymorphisms revealed a higher OR for TP53 72Pro carriers homozygous for p21 31Ser than for 72Pro carriers in general; this effect being most pronounced in heavy smokers with squamous-cell carcinoma (OR, 3.84; CI, 1.46-10.1). Our data indicate that the TP53 Arg72Pro polymorphism increases the risk for squamous-cell carcinoma mainly in heavy smokers. The observed interaction with smoking is biologically plausible as, for the 72Pro p53 variant, decreased apoptosis and extended G1 cell cycle arrest is reported after carcinogen exposure. Nevertheless, confirmation by further molecular and epidemiological studies is warranted.

Correlation of cyclin D1 and E1 expression with bladder cancer presence, invasion, progression, and metastasis

The objective of this study was to determine the correlation of the expression of cyclin D1 and E1 with the expression of commonly altered cell cycle regulators and bladder cancer presence, staging, and clinical outcomes. We performed immunohistochemical staining for cyclin D1, cyclin E1, p53, p21, p27, and retinoblastoma protein (pRB) on serial cuts from normal urothelium from 9 controls, radical cystectomy specimens from 226 consecutive patients with advanced transitional cell carcinoma, and lymph nodes with metastasis from 50 of the 226 cystectomy patients. Cyclin D1 and E1 immunoreactivity were considered low when samples demonstrated less than 10% and 30% nuclear reactivity, respectively. Normal bladder urothelium from all 9 control patients showed uniformly intense expression of cyclin D1 and E1. Cyclin D1 expression was low in 99 (43.8%) of 226 cystectomy specimens and 25 (50.0%) of 50 metastatic lymph node specimens. Cyclin D1 immunoreactivity was not associated with any pathologic characteristics or clinical outcomes. Cyclin E1 expression was low in 125 (55.3%) of 226 cystectomy specimens and 22 (44.0%) of 50 metastatic lymph node specimens. Low cyclin E1 expression was significantly associated with advanced pathologic stage, lymphovascular invasion, and lymph node metastases. In multivariate analyses, low cyclin E1 expression was significantly associated with bladder cancer-specific mortality (P = .048), but not disease recurrence (P = .056). Low cyclin E1 expression was significantly associated with altered expression of pRB, p27, and cyclin D1. Low cyclin D1 expression was significantly associated with altered expression of pRB, p21, and cyclin E1. Cyclin E1 expression stratifies patients with bladder transitional cell carcinoma into those with more "indolent" behavior and those with features of biologically and clinically aggressive disease.

Altered gene expression induced by ionizing radiation and glycolytic inhibitor 2-deoxy-glucose in a human glioma cell line: Implications for radio sensitization

The glycolytic inhibitor 2-deoxy-D-glucose (2DG) has been shown to enhance the cell death induced by radiation and other DNA damaging agents selectively in cells with high rates of glycolysis, like cancer cells. While energy linked modification of DNA and cellular repair processes have been suggested as possible mechanisms of sensitization, other effects such as global stress response cannot be excluded. In this pilot study, we have investigated the effect of 2-DG and radiation on the transcriptome in an attempt to elucidate how 2-DG impacts gene expression in undamaged verses irradiation (IR) damaged cells using a human malignant glioma cell line, U-87. Exponentially growing U-87 cells were exposed to various combinations of 2-DG and X-rays and total RNA was isolated four hours after exposure. Gene expression changes were elucidated using Affymetrix GeneChips. As expected, U-87 cells treated with 2-DG showed activation of several endoplasmic reticulum stress response genes. Selective RT-PCR and western blotting confirmed these gene alterations. Given that glucose deprivation leads to p53 activation and 2-DG led to activation of p53 response genes in our present study (e.g. PMAIP1 and GADD45A), we examined the impact of transient p53 knockdown and observed that induction of PMAIP1 and GADD45A appear to be via p53-independent mechanisms. The majority of gene alterations seen with IR-treatment alone were consistent with previous reports. While most gene alterations seen with 2-DG and IR dual treatment were confirmed in the gene profiles seen with individual (2-DG or IR) treatments, several genes appeared differentially regulated between IR and 2-DG (e.g. DUSP8, IL8, GADD45B). Additionally, gene expression patterns suggested alterations in cell cycle regulation, apoptosis, and cytokine signaling pathways. Taken together, this study provides new insights into how the transcriptome of tumor cells are likely to be affected by a combined stress caused by IR and 2-DG.

Altered expression of cell cycle regulatory proteins in gastrointestinal stromal tumors: markers with potential prognostic implications

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the digestive tract. The prediction of the malignant potential of GISTs is still difficult. Altered cell cycle regulation may underlie the tumorigenesis and/or the progression of human malignancies. Although p53 and Bcl-2 have been extensively investigated in GISTs, little is known about the frequency of expression and possible clinical implications of alterations of other cell cycle regulatory proteins in these neoplasms. We have previously investigated the role of loss of p16(INK4A) by loss of heterozygosity and immunohistochemistry in the progression of GISTs and found that loss of heterozygosity of 9p and loss of p16 expression are confined to malignant GISTs. This has led us to investigate the role of other cell cycle regulatory proteins in these tumors. Twenty-three cases of GIST (9 low malignant potential [LMP], 10 primary malignant, and 4 intra-abdominal recurrences) were examined. All cases were strongly positive for KIT (CD117). Immunohistochemical stains were carried out on tissue microarrays to evaluate the expression of proteins involved in the G(1)-S transition and proteins that regulate apoptosis including Rb, E2F1, cyclin D1, CDK4, CDK6, p27(KIP1), p21(WAF1/CIP1), p53, Mdm2, Bcl-2, and Bax. The positive phenotypes identified were as follows: Rb, 39.1%; E2F1, 69.6%; cyclin D1, 30.4%; CDK4, 100%; CDK6, 30.4%; 39.1%; p27(KIP1), 47.8%; p21(WAF1/CIP1), 39.1%; p53, 43.5%; Mdm2, 17.4%; Bcl-2, 91.3%; and Bax, 100%. Malignant GISTs are more likely to be associated with a positive E2F1 and p53 phenotype and a negative p16 and p27(KIP1) phenotype. It was concluded that aberration of the cell cycle regulators is a frequent finding and may be a contributing factor to the pathogenesis of GISTs. While some alterations are seen in LMP and malignant GISTs and therefore may represent an early event in molecular tumorigenesis of GISTs, other alterations are more common in malignant GISTs than LMP and therefore have potential utility as complementary tools for the prognostication of GISTs.

TGF-β signaling pathway inactivation and cell cycle deregulation in the development of gastric cancer: Role of the β-spectrin, ELF

We have shown that loss of ELF, a stem cell adaptor protein, disrupts TGF-β signaling through Smad3 and Smad4 localization. Notably elf +/− /smad4 +/− mice develop gastric cancer presenting this as an important model for analyzing molecular event in gastric carcinogenesis. To gain further insight into the functional role of ELF in gastric cancer suppression, we carried out a detailed characterization of cell cycle events leading to gastric tumorigenesis. elf −/− cells and elf +/− /smad4 +/− mice demonstrate a marked alteration of cell cycle regulators, such as Cdk4, K-Ras, and p21. Levels of Cdk4 increased compared to normal controls, suggesting loss of ELF results in functional abnormalities in cell cycle regulation. We further demonstrate that the elf −/− MEFs show a disruption of G 1/S cell cycle transition and a significant reduction in senescence. Thus, in response to ELF deficiency, the abnormalities of G 1/S checkpoint and senescence contribute their increment of susceptibility to malignant transformation.

P27Kip1 is Detected on Most Gastric MALT Lymphomas, but not Large Cell Lymphomas

We investigated the relationship of gastric mucosa-associated lymphoid tissue (MALT) lymphoma tumorigenesis to Helicobacter pylori infection, the t (11;18) translocation, and alterations in cell cycle regulators. We sought to assess the implications of altered expression of p27(Kip1), a cyclin-dependent kinase inhibitor, on high-grade transformation and responsiveness to eradication therapy. We used immunohistochemistry to examine p27(Kip1), p53, and Ki-67 expression in 23 MALT lymphomas, five diffuse large B-cell lymphomas (DLBCLs), and four DLBCLs with associated MALT lymphoma. All of the MALT lymphomas were positive for p27(Kip1) expression and negative for p53 with a low Ki-67 index, regardless of the sensitivity of these cells to eradication. All DLBCLs were negative for p27(Kip1) and positive for p53, exhibiting a high Ki-67 index. In DLBCLs with MALT lymphoma, p27(Kip1) expression was absent from both the MALT and large cells components. In all of these lymphomas, the MALT components were negative for p53 and displayed a low Ki-67 index, while the large cell components were positive for p53 with a high Ki-67 index. The expression patterns of the DLBCLs differed significantly from those of the MALT lymphomas. p27(Kip1) was not detected in either component of DLBCL with MALT lymphoma, suggesting that decreased expression of p27(Kip1) in the MALT component may be related to high-grade transformation. Thus, p27(Kip1) expression in morphological MALT lymphomas could be useful tool to predict high-grade transformation to DLBCL.

Inhibition of Proteasome Is Associated with Early Alterations of Cell Cycle Regulators and Induces Synergistic Antitumour Activities in Mantle Cell Lymphoma in a Sequence Dependent Manner.

Mantle cell lymphoma (MCL) displays an especially poor clinical outcome with only transient response to conventional chemotherapy, continuous relapses and a median survival of only 3 - 4 years. The Ubiquitin-proteasome pathway is known to alter homeostasis of various oncogenes, transcription factors (e.g. NF-κB) regulators of cell cycle progression and apoptosis. In various phase II trials, proteasome inhibition with single agent bortezomib (Velcade®) achieved response rates of up to 60% in relapsed disease. Five MCL cell lines (HBL2, Granta 519, Jeko-1, NCEB-1 and Rec-1) and two hematological control cell lines (Jurkat, Karpas 422) were treated with bortezomib at a previously defined effective concentration (25 nM). Real-time RT-PCR and Western Blot analysis of cyclin D1 (CCND1), Cdk inhibitors (INK4s, KIPs), and other regulators of cell cycle and apoptosis were performed at various time points during bortezomib treatment (0 to 12 hours). Additionally, analyses of cell cycle were performed by flow cytometry. All cells lines were also exposed to different doses of bortezomib in combination with various cytostatic drugs, cell proliferation (WST-1 assay) and apoptosis (Annexin V PE/7-AAD staining) were analysed. After only 2 - 4 hours of bortezomib treatment analysis of relative RNA expression levels revealed downregulation of Cdk4 inhibitor p21CIP1, CCND1 and BCL2, thus representing early effects of proteasome inhibition in MCL cell lines. In contrast, CCND1 expression temporarily increased in cell lines with moderate sensitivity to bortezomib. Interaction between bortezomib and cytostatic drugs were evaluated applying the combination index (CI). Simultaneous exposure to bortezomib and various cytostatic drugs (cytarabine, mitoxantrone, fludarabine and gemcitabine) resulted in a significantly enhanced inhibition of proliferation and apoptosis. In order to investigate the interaction of cytostatic agents and bortezomib different incubation schedules were evelusated. Notably, pre-exposure to cytarabine and subsequent proteasome inhibition results in a strong synergism (CI = 0.5) that was not observed with prior bortezomib incubation. Interesting this pattern is chemotherapy specific, since bortezomib pre-incubation induced synergism with mitoxantrone (CI = 0.6). Finally, combination of cytarabine or gemcitabine with bortezomib was able to sensitize and inhibit cell proliferation of MCL cell lines resistant to single agent treatment. In summary, alteration of protein expression profiles and cell cycle regulators occur early after proteasome inhibition in MCL cell lines. In addition, combination of bortezomib with distinct cytostatic agents demonstrated a synergistic schedule dependent efficacy in vitro. Currently, confirmatory analyses of primary patient samples are being performed representing the rationale of a future randomized phase II/III study of the European MCL Network.

The epigenome as a molecular marker and target

Tumor cell proliferation, de-differentiation, and progression depend on a complex combination of altered cell cycle regulation, excessive growth factor pathway activation, and decreased apoptosis. The understanding of these complex mechanisms should lead to the identification of potential molecular markers, targets, and molecular profiles that should eventually expand and improve therapeutic intervention. It now appears clear that methylation plays a central role in transformation, both in vitro and in vivo. However, the exact targets and mechanism(s) are not yet fully understood. This is partly due to the significant number of genes altered by changes in intracellular methyltransferase activity and the chemical agents used to modulate gene expression. The complex nature of methylation's role in regulating gene expression suggests that in addition to investigating individual genes, researchers should develop more comprehensive methods to examine gene expression patterns and their predictive value as this will likely be necessary in the future. If methylation plays a role in transformation, then it seems logical that genes regulating intracellular methylation status may be used as molecular markers to profile tumors by any new methods currently being developed. Perhaps more noteworthy is that DNMT genes may be found to be novel molecular targets for new factor-specific anticancer agents. This idea will be addressed.

Prognostic significance of expressions of cell-cycle regulatory proteins in gastrointestinal stromal tumor and the relevance of the risk grade

Gastrointestinal stromal tumors (GISTs) have a wide spectrum of biologic behavior ranging from benign to malignant. Risk grading based on tumor size and mitotic counts has been proposed in an effort to predict the adverse outcome of GIST in the literature so far. Recent molecular studies have reported the prognostic values of several parameters, including alteration of cell-cycle regulators. The aim of this study was to elucidate the prognostic values of risk grade and alterations of cell-cycle-related proteins, including Ki-67, cyclin A, cyclin B1, cyclin D1, cyclin E, p16, p21, p27, p53, cdc2, and cdk2, in addition to the conventional factors. Eighty cases of primary c-kit-positive GISTs were classified into 2 cases of very-low-risk grade, 20 cases of low-risk grade, 25 cases of intermediate-risk grade, and 33 cases of high-risk grade. The risk grade was correlated with the presence of metastases and/or recurrence. A high level of Ki-67 and cyclin A expression was correlated with risk grade (P = .0027 and .0441, respectively). Overexpression of G2-M regulators, such as cyclin A, cyclin B1, and cdc2, was associated with the Ki-67 labeling index (LI) (P = .0007, .0475, and .0040, respectively). According to univariate analysis, tumor grade (high risk), tumor size (> or =5 cm), mitotic counts (> or =5/50 high-power fields), Ki-67 LI (> or =4.92%), cyclin A LI (> or =1.61%), and cdc2 LI (> or =1.25%) were all found to be significantly associated with a shorter period of disease-free survival (P = .0001, .0270, .0004, .0001, .0001, and .0011, respectively). According to multivariate analysis, both high Ki-67 LI and high-risk grade were found to be significantly associated with a shorter period of disease-free survival (P = .0083 and .0246, respectively). In conclusion, our results strongly support the hypothesis that Ki-67 LI and risk grade are useful for predicting the aggressive biologic behavior of GISTs. Furthermore, alteration of G2-M regulators, such as cyclin A, cyclin B1, and cdc2, is also a useful marker for predicting aggressive behavior and play an important role, at least in part, in the cell proliferation of GIST.

Thioredoxin reductase as a novel molecular target for cancer therapy

Tumor cell proliferation, de-differentiation, and progression depend on a complex combination of altered cell cycle regulation, excessive growth factor pathway activation, and decreased apoptosis. The understanding of these complex mechanisms should lead to the identification of potential targets for therapeutic intervention. Redox-sensitive signaling factors also regulate multiple cellular processes including proliferation, cell cycle, and pro-survival signaling cascades, suggesting their potential as molecular targets for anticancer agents. These observations suggest that redox-sensitive signaling factors may be potential novel molecular markers. We hypothesized that thioredoxin reductase-1 (TR), a component of several redox-regulated pathways, may represent a potential molecular target candidate in response to agents that induce oxidative stress. There have been numerous biological studies over the last decade investigating the cell biological, biochemical, and genetic properties of TR both in culture and in in vivo models. In addition, using a series of permanent cell lines that express either a wild-type TR or a dominant mutant TR gene or a chemical agent that inhibits TR we demonstrated that TR meets most criteria that would identify a molecular target. Based on these results we believe TR is a potential molecular target and discuss potential clinical possibilities.

The proteasome inhibitor bortezomib induces rapid alterations of cell cycle regulators and augments sensitivity to cytostatic drugs in mantle cell lymphoma

6609 Background: Mantle cell Lymphoma (MCL) is a distinct subtype of malignant lymphoma with an especially poor clinical outcome and only transient response to chemotherapy. In various phase II studies the proteasome inhibitor bortezomib (Velcade) achieved remarkable response rates of up to 60% in relapsed MCL. However, besides NF-κB, little is known about the critical target molecules mainly affected by proteasome inhibition. Methods: Four MCL cell lines (HBL2, Granta 519, Jeko-1, NCEB-1) and two hematological control cell lines (Jurkat, Karpas 422) were exposed to bortezomib at a previously defined cytotoxic concentration (25 nmol). Real-time RQ-PCR and Western Blot analysis of cyclin D1, various cdk inhibitors (INK4s, KIPs), and other regulators of cell cycle and apoptosis were performed at various time points during bortezomib treatment (0,5 - 12 hours). In addition, cell cycle phase and apoptosis (Annexin V PE/7-AAD staining) were directly determined by flow cytometry. All cells lines were also simultaneously exposed to different doses of bortezomib and various cytostatic drugs (WST1 assay). Results: Analysis of relative RNA expression levels revealed that downregulation of cdk4 inhibitors was observed already after a few hours of bortezomib treatment and thus represents an early effect of proteasome inhibition in MCL cell lines. In contrast, cyclin D1 expression temporarily increased especially in cell lines with only moderate sensitivity to bortezomib. Interestingly, efficacy of proteosome inhibition varied widely between the MCL cell lines. Simultaneous exposure of bortezomib and various cytostatic drugs (especially cytarabine) resulted in a significant inhibition of proliferation and enhanced apoptosis. Conclusions: In summary, alteration of cell cycle regulators represents an early effect of proteasome inhibition in MCL. In addition, a superadditive efficacy of bortezomib and distinct cytostatic agents was observed in the cell line model. Currently, confirmatory analyses of primary patient samples are being performed representing the rationale of a future randomized phase II/III study of the European MCL Network. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ortho-Biotech

Rapamycin inhibits human in stent restenosis vascular smooth muscle cells independently of pRB phosphorylation and p53

Drug-eluting stents containing the immunosuppressant rapamycin markedly inhibit in stent restenosis (ISR). However, the molecular mechanisms that underlie its effect on ISR-derived vascular smooth muscle cells (VSMCs), as opposed to normal VSMCs, are unknown. Specifically, as ISR-VSMCs have altered cell cycle regulation, rapamycin may arrest these cells via novel molecular pathways. We isolated human VSMCs from sites of ISR, and examined the effect of rapamycin on cell proliferation using MTT assay, time lapse videomicroscopy and flow cytometry. Regulation of G(1)-S transition was examined using Western blotting, and cell size and protein synthesis examined using flow cytometry and collagen assay, respectively. The requirement for pRB and p53 was examined using ISR VSMCs expressing E1A and a dominant negative p53, respectively. ISR-VSMC proliferation was potently inhibited by rapamycin. Arrest was confined to G(1), as cell proliferation (but not cell size) of S/G(2)-arrested cells was unaffected by rapamycin. Moreover, ISR-VSMC lines generated with disrupted p53 or pRB function still arrested in the presence of rapamycin, suggesting that these genes are dispensable for rapamycin-induced arrest. Significantly, rapamycin completely inhibited the phosphorylation of p70(S6K), an mTOR-regulated kinase implicated in the control of proliferation, but had no effect on collagen or total protein synthesis. We demonstrate that rapamycin is a potent inhibitor of ISR VSMC proliferation during G(1). Rapamycin's action does not require p53 or pRB. We show that p70(S6K) is markedly inhibited in rapamycin-arrested ISR cells, suggesting that regulation of its upstream kinase, mTOR, is important for the control of proliferation in ISR cells.

Gene Expression in Normal Human Bronchial Epithelial (NHBE) Cells Following In Vitro Exposure to Cigarette Smoke Condensate

Cigarettes that burn tobacco produce a complex mixture of chemicals, including mutagens and carcinogens. Cigarettes that primarily heat tobacco produce smoke with marked reductions in the amount of mutagens and carcinogens and demonstrate reduced mutagenicity and carcinogenicity in a battery of toxicological assays. Chemically induced oxidative stress, DNA damage, and inflammation may alter cell cycle regulation and are important biological events in the carcinogenic process. The objective of this study was to characterize and compare the effects of smoke condensates from cigarettes that burn tobacco and those that primarily heat tobacco on gene expression in NHBE cells. For this comparison, we used quantitative RT/PCR and further evaluated the effects on cell cycling using flow cytometry. Cigarette smoke condensates (CSCs) were prepared from Kentucky 1R4F cigarettes (a tobacco-burning product designed to represent the average full-flavor, low "tar" cigarette in the US market) and Eclipse (a cigarette that primarily heats tobacco) using FTC machine smoking conditions. The CSC from 1R4F cigarettes induced statistically significant increases in the mRNA levels of genes responsive to DNA damage (GADD45) and involved in cell cycle regulation (p21;WAF1/CIP1), compared to the CSC from Eclipse cigarettes. In addition, genes coding for cyclooxygenase-2 (COX-2) and interleukin 8 (IL-8), which are associated with oxidative stress and inflammation, respectively, were increased statistically significantly more by CSC from 1R4F than by that from Eclipse. Furthermore, a dose-dependent increase in IL-8 protein secretion into cell culture media was stimulated by 1R4F exposure, whereas minimal IL-8 protein was secreted after Eclipse treatment. The biological relevance of the differential effect on gene expression was reflected in differential cell cycle regulation, as cells exposed to 1R4F CSC exhibited more significant S phase and G2 phase accumulation than cells exposed to Eclipse CSC. These data indicate that the simplified smoke chemistry of the tobacco-heating Eclipse cigarette yields statistically significant reductions in the expression of key genes involved in DNA damage, oxidative stress, inflammatory response, and cell cycle regulation in normal human bronchial epithelial cells compared to a representative tobacco-burning cigarette.

Alterations of cell cycle regulators in gliomatosis cerebri

Gliomatosis cerebri (GC) is regarded as a rare glial neoplasm of unknown origin, and a detailed analysis of molecular alterations underlying this disease has started only recently. However, because GC characteristically affects large parts of the brain and spinal cord, the distribution of genetic alterations may be highly variable between different tumor areas. Additionally, tumor areas with varying degrees of differentiation may be present, raising the possibility to model the genetic events associated with astrocytoma progression. Here we analyzed various tumor regions with features of low-grade and high-grade astrocytomas from 9 autopsy-proven GC cases for the immunoexpression of the cell cycle-controlling proteins mdm2, p21, p27/kip1, p16, and Rb. The samples were also screened for EGFR expression, and for amplification of the EGFR and MDM2 genes. Furthermore, allelic losses of the CDKN2A gene and of a PTEN flanking region of chromosome 10 were determined. We detected tumor regions with immunoexpression of p21 only rarely in our series, without association to the tumor grade. No MDM2 gene amplification was detected. In contrast, three cases demonstrated maintained Rb expression. The expression of p27(kip1) showed a clear reduction with increasing astrocytoma malignancy in 7 cases. Allelic loss of the CDKN2A gene occurred in 5 patients but was not related to the tumor grading, nor to the intensity of p16 immunoexpression. No homozygous CDKN2Adeletions were detected. EGFR amplification was also absent in our series, but one case demonstrated EGFR expression only in the high-grade tumor area. Allelic losses on chromosome 10 were found in one out of six informative cases. However, marked differences in the immunoexpression, as well as in the distribution of genetic aberrations were seen between different tumor samples within a given case. The distribution of the alterations suggests that these molecular genetic changes represent secondary events, which may develop within tumor clones derived from a common founder tumor clone characterized by extraordinary spreading through the brain. Moreover, the detected aberrations in gliomatosis cerebri can reflect the tumor progression associated with secondary malignant astrocytoma formation even within a single case.

Alteration ofAKAP220, an upstream component of the Rb pathway, in oral carcinogenesis

Few genes have been implicated in the development of oral cancer. In our study, we identified a novel gene in the Rb pathway that is frequently altered and overexpressed in oral tumors. Significantly, the alteration is also associated with early oral premalignant lesions (OPLs). This region was identified through a genomewide scan using randomly amplified polymorphic DNA (RAPD) PCR of 40 microdissected oral squamous cell carcinomas (SCCs). Recurrent gain of a approximately 400 bp signal was observed in multiple patients. This gain was localized to 13q14.11, a region frequently altered in multiple cancer types. Through microsatellite analysis, a 1.9 Mbp minimal region of alteration (MRA) was defined between D13S263 and D13S1227. Allelic imbalance (AI) in the MRA was present in only 28% of low-grade dysplasia, but strikingly increased with progression to 64% in high-grade dysplasia, plateauing at 61% in tumors, thus implicating this alteration in the early stages of disease development. Of the 3 genes residing within the MRA, Receptor Activator of NK-kappa-B Ligand (RANKL) and Diacylglycerol Kinase (DGKH) showed no change in expression levels in tumors compared to normal tissue. In contrast, 12 of 16 tumors showed significant overexpression of A-Kinase Anchoring Protein 220 (AKAP220). Since AKAP220 plays a role in regulating the Rb pathway, its dysregulation may contribute significantly to alterations in cell cycle regulation that facilitate progression of OPLs.