Malignant transformation in human chondrosarcoma cells supported by telomerase activation and tumor suppressor inactivation.

Human chondrosarcomas do not respond to current chemotherapies or radiation therapy, and their size and histological appearance do not reliably predict the risk of local recurrence and metastases, making selection of surgical treatment difficult. Identifying mechanisms responsible for the proliferation and invasive behavior of these tumors would be of immense clinical value. We hypothesized that telomerase expression is one of these mechanisms. We detected telomerase expression in 7 of 16 chondrosarcomas, but cells cultured from telomerase-negative chondrosarcomas acquired strong telomerase activity and lost tumor suppressor activity after their establishment in culture. These changes were associated with accelerated indefinite cell proliferation, morphological transition, and increased invasive activity, indicating that telomerase activation and loss of cell cycle control leads to the emergence of aggressive cells from chondrosarcoma cell populations. These observations may lead to better understanding of the factors responsible for malignant transformation, local recurrence, and metastases of cartilage neoplasms.     

Compensation of BRG-1 function by Brm: insight into the role of the core SWI-SNF subunits in retinoblastoma tumor suppressor signaling.

The BRG-1 subunit of the SWI-SNF complex is involved in chromatin remodeling and has been implicated in the action of the retinoblastoma tumor suppressor (RB). Given the importance of BRG-1 in RB function, germ line BRG-1 mutations in tumorigenesis may be tantamount to RB inactivation. Therefore, in this study we assessed the behavior of cells harboring discrete BRG-1 alleles for the RB-signaling pathway. Using p16ink4a, an upstream activator of endogenous RB, or a constitutively active RB construct (PSM-RB), we determined that the majority of tumor lines with germ line defects in BRG-1 were sensitive to RB-mediated cell cycle arrest. By contrast, A427 (lung carcinoma) cells were resistant to expression of p16ink4a and PSM-RB. Analysis of the SWI-SNF subunits in the different tumor lines revealed that A427 are deficient for BRG-1 and its homologue, Brm, whereas RB-sensitive cell lines retained Brm expression. Similarly, the RB-resistant SW13 and C33A cell lines were also deficient for both BRG-1/Brm. Reintroduction of either BRG-1 or Brm into A427 or C33A cells restored RB-mediated signaling to cyclin A to cause cell cycle arrest. Consistent with this compensatory role, we observed that Brm could also drive expression of CD44. We also determined that loss of these core SWI-SNF subunits renders SW13 cells resistant to activation of the RB pathway by the chemotherapeutic agent cisplatin, since reintroduction of either BRG-1 or Brm into SW13 cells restored the cisplatin DNA-damage checkpoint. Together, these data demonstrate that Brm can compensate for BRG-1 loss as pertains to RB sensitivity.     

Stabilization of the retinoblastoma protein by A-type nuclear lamins is required for INK4A-mediated cell cycle arrest

Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16(ink4a), we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16(ink4a)-mediated G(1) arrest. Reintroduction of lamin A, lamin C, or pRB restores p16(ink4a)-responsiveness to Lmna(-/-) cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna(-/-) cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16(ink4a) responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.     

p53‐dependent G1 arrest in 1st or 2nd cell cycle may protect human cancer cells from cell death after treatment with ionizing radiation and Chk1 inhibitors

Objectives: This study was performed to explore the strategy of combining Chk1 inhibitors with ionizing radiation (IR) to selectively target p53‐deficient cancer cells. Materials and methods: Survival and cell cycle progression were measured in response to IR and the Chk1 inhibitors, UCN‐01 and CEP‐3891, in colon carcinoma HCT116 p53+/+ and p53?/? cells, and in osteosarcoma U2OS‐VP16 cells with conditional expression of dominant‐negative p53 (p53DD). Results: Clonogenic survival was selectively reduced in HCT116 p53?/? compared to p53+/+ cells after treatment with UCN‐01 and IR, and HCT116 p53+/+ cells also displayed strong p53‐dependent G₁ arrest in the 1st cell cycle after IR. In contrast, clonogenic survival was affected similarly in U2OS‐VP16 cells with and without expression of p53DD. However, death of U2OS‐VP16 cells was p53 dependent as assessed by cell viability assay at 72 h, and this was associated with p53‐dependent G₁ arrest in the 2nd cell cycle after treatment. Notably, HCT116 cells were overall more resistant than U2OS cells to cytotoxic effects of Chk1 inhibitors. Conclusion: Our results suggest that p53‐dependent G₁ arrest in both 1st and 2nd cell cycles may protect human cancer cells from cell death after treatment with IR and Chk1 inhibitors. However, a challenge for future clinical use will be that different cancers display different intrinsic sensitivity to such inhibitors.     

Cyclin-dependent Kinases Participate in Death of Neurons Evoked by DNA-damaging Agents

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus–induced expression of CKIs p16^ink4, p21^waf/cip1, and p27^kip1, as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation– and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1–associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.     

Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of “stemness.” This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence.     

Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.     

Proteomic analysis of p16ink4a-binding proteins

The p16(ink4a) tumor suppressor protein plays a critical role in cell cycle control, tumorogenesis and senescence. The best known activity for p16(ink4a) is the inhibition of the activity of CDK4 and CDK6 kinases, both playing a key role in cell cycle progression. With the aim to study new p16(ink4a) functions we used affinity chromatography and MS techniques to identify new p16(ink4a)-interacting proteins. We generated p16(ink4a) columns by coupling the protein to activated Sepharose 4B. The proteins from MOLT-4 cell line that bind to p16(ink4a) affinity columns were resolved by SDS-PAGE and identified by MS using a MALDI-TOF. Thirty-one p16(ink4a) -interacting proteins were identified and grouped in functional clusters. The identification of two of them, proliferating cell nuclear antigen (PCNA) and minichromosome maintenance protein 6 (MCM6), was confirmed by Western blotting and their in vivo interactions with p16(ink4a) were demonstrated by immunoprecipitation and immunofluorescence studies. Results also revealed that p16(ink4a) interacts directly with the DNA polymerase delta accessory protein PCNA and thereby inhibits the polymerase activity.     

Epigallocatechin-3-gallate induces cell apoptosis of human chondrosarcoma cells through apoptosis signal-regulating kinase 1 pathway

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. The aim of this study was to elucidate the mechanism of EGCG-induced apoptosis of human chondrosarcoma cells. EGCG induced cell apoptosis in human chondrosarcoma cell lines but not primary chondrocytes. EGCG induced upregulation of Bax and Bak, downregulation of Bcl-2 and Bcl-XL, and dysfunction of mitochondria in chondrosarcoma. We also found that the accumulation of reactive oxygen species (ROS) is a critical mediator in EGCG-induced cell death. EGCG induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of chondrosarcoma cells with EGCG induced p38 and c-jun-NH2-kinase (JNK) phosphorylation. Transfection with ASK1 siRNA or p38 and JNK mutant antagonized the EGCG-induced cell apoptosis. Therefore, EGCG triggered ROS and activated the ASK1-p38/JNK pathway, resulting chondrosarcoma cell death. Importantly, animal studies revealed a dramatic reduction in tumor volume after 24 days of treatment. Thus, EGCG may be a novel anti-cancer agent for the treatment of chondrosarcoma.     

5-Bromodeoxyuridine induces senescence-like phenomena in mammalian cells regardless of cell type or species

5-Bromodeoxyuridine was found to induce flat and enlarged cell shape, characteristics of senescent cells, and senescence-associated beta-galactosidase in mammalian cells regardless of cell type or species. In immortal human cells, fibronectin, collagenase I, and p21(wafl/sdi-1) mRNAs were immediately and very strongly induced, and the mortality marker mortalin changed to the mortal type from the immortal type. Human cell lines lacking functional p21(wafl/sdi-1), p16(ink4a), or p53 behaved similarly. The protein levels of p16(ink4a) and p53 did not change uniformly, while the level of p21(wafl/sdi-1) was increased by varying degrees in positive cell lines. Telomerase activity was suppressed in positive cell lines, but accelerated telomere shortening was not observed in tumor cell lines. These results suggest that 5-bromodeoxyuridine activates a common senescence pathway present in both mortal and immortal mammalian cells.     

3-Deazaneplanocin A (DZNep), an Inhibitor of the Histone Methyltransferase EZH2, Induces Apoptosis and Reduces Cell Migration in Chondrosarcoma Cells

Objective

Growing evidences indicate that the histone methyltransferase EZH2 (enhancer of zeste homolog 2) may be an appropriate therapeutic target in some tumors. Indeed, a high expression of EZH2 is correlated with poor prognosis and metastasis in many cancers. In addition, 3-Deazaneplanocin A (DZNep), an S-adenosyl-L homocysteine hydrolase inhibitor which induces EZH2 protein depletion, leads to cell death in several cancers and tumors. The aim of this study was to determine whether an epigenetic therapy targeting EZH2 with DZNep may be also efficient to treat chondrosarcomas.

Methods

EZH2 expression was determined by immunohistochemistry and western-blot. Chondrosarcoma cell line CH2879 was cultured in the presence of DZNep, and its growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7 expression using flow cytometry, and by PARP cleavage using western-blot. Cell migration was assessed by wound healing assay.

Results

Chondrosarcomas (at least with high grade) highly express EZH2, at contrary to enchondromas or chondrocytes. In vitro, DZNep inhibits EZH2 protein expression, and subsequently reduces the trimethylation of lysine 27 on histone H3 (H3K27me3). Interestingly, DZNep induces cell death of chondrosarcoma cell lines by apoptosis, while it slightly reduces growth of normal chondrocytes. In addition, DZNep reduces cell migration.

Conclusion

These results indicate that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treat chondrosarcomas.     

FK506 confers chemosensitivity to anticancer drugs in glioblastoma multiforme cells by decreasing the expression of the multiple resistance-associated protein-1

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstream of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC₅₀ values 0.01 to 0.6 μM) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r = 0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p < 0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.     

Induction of apoptosis in p16INK4A mutant cell lines by adenovirus-mediated overexpression of p16INK4A protein

The tumor suppressor gene p16INK4A is a cyclin-dependent kinase inhibitor (CDKI) and an important cell cycle regulator. We have previously constructed a recombinant adenovirus which expresses p16 (Adp16) and shown that infection in a variety of human tumor cell lines with this recombinant virus results in high levels of p16INK4A protein expression resulting in cell cycle arrest and loss of cyclin-cdk activity. Furthermore, adenoviral-mediated overexpression of wild-type p16INK4A is more toxic in cancer cells which express mutant forms of p16INK4A compared to cancer cell lines containing endogenous wild-type p16. TUNEL assay and DAPI staining following infection of MDA-MB 231 breast cancer cells with Adp16 indicate that p16INK4A-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating a decrease in cpp32 and cyclinB1 protein levels and induction of poly (ADP-ribose) polymerase (PARP) cleavage following infection of MDA-MB-231 cells with Adp16. These results suggest that gene therapy using Adp16 may be a promising treatment option for human cancers containing alterations in p16 expression.     

Expression of apoptosis and cell cycle related genes in proliferating and colcemid arrested cells of divergent lineage.

Progression through the cell cycle and redirection of cells towards programmed cell death (apoptosis) are tightly inter-related processes. However the requirement for tissue and cell type specificity suggests that a wide variety of mechanisms are used to achieve the same purpose. To examine this issue, we investigated cell cycle (c-myc, p53, p21/WAF) and apoptosis related (bcl-2, bcl-X(L), bax-alpha) gene expression in two cell lines of very different origin under proliferating and apoptosis-inducing conditions. Transformed human osteosarcoma cells (MG63) and non-transformed human kidney embryonal fibroblasts (293-0) were kept in culture in medium containing 10% FCS and growth arrest was induced by the addition of 50 ng/ml colcemid. Colcemid treatment caused growth arrest and elevated expression of cyclin B1 protein in both cell lines. Apoptosis was significantly elevated in both cell lines after colcemid exposure for at least one cell cycle. However the pattern of expression of cell cycle and apoptosis related genes, determined by RT-PCR, was quite different between the two cell lines during exponential growth and cell cycle arrest. Colcemid treatment did not markedly influence c-myc, p53 and p21/WAF expression in MG63 cells but did suppress c-myc and increase p21/WAF in 293-0 cells. Furthermore colcemid treated MG63 cells exhibited elevated bcl-2 and bax-alpha expression while similar treatment of 293-0 cells resulted in decreased bcl-X(L) and slightly increased bax-alpha expression. While growth arrest and apoptosis were induced in both MG63 and 293 cells following colcemid treatment, the differences in gene expression suggest that the mechanism by which these cells determine cell fate is quite different and may determine the sensitivity of different cell populations to anti-neoplastic drug therapy. The distinct patterns of gene expression should be carefully defined before mechanisms of apoptotic cell death are studied.     

Paradoxical effects of a stress signal on pro- and anti-apoptotic machinery in HTLV-1 tax expressing cells

Adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) are associated with Human T-cell lymphotropic virus type 1 (HTLV-1) infection. The viral transactivator, Tax is able to mediate the cell cycle progression by targeting key regulators of the cell cycle such as p21/waf1, p16/ink4a, p53, cyclins D1-3/cdk complexes, and the mitotic spindle checkpoint MAD apparatus, thereby deregulating cellular DNA damage and checkpoint control. Genome expression profiling of infected cells exemplified by the development of DNA microarrays represents a major advance in genome-wide functional analysis. Utilizing cDNA microarray analysis, we have observed an apparent opposing and paradoxical regulatory network of host cell gene expression upon the introduction of DNA damage stress signal. We find the apparent induction of cell cycle inhibitors, and pro- as well as anti-apoptotic gene expression is directly linked to whether cells are at either G1, S, or G2/M phases of the cell cycle. Specifically, a G1/S block is induced by p21/waf1 and p16/ink4a, while pro-apoptotic expression at S, and G2/M is associated with caspase activation, and anti-apoptotic gene expression is associated with up regulation of Bcl-2 family member, namely bfl-1 gene. Therefore, the microarray results indicating expression of both pro- and anti-apoptotic genes could easily be explained by the particular stage of the cell cycle. Mechanism and the functional outcome of induction for both pathways are discussed.     

Downregulation of CXXC Finger Protein 4 Leads to a Tamoxifen-resistant Phenotype in Breast Cancer Cells Through Activation of the Wnt/β-catenin Pathway

Tamoxifen is a successful endocrine therapy drug for estrogen receptor–positive (ER+) breast cancer. However, resistance to tamoxifen compromises the efficacy of endocrine treatment. In the present study, we identified potential tamoxifen resistance–related gene markers and investigated their mechanistic details. First, we established two ER + breast cancer cell lines resistant to tamoxifen, named MCF-7/TMR and BT474/TMR. Gene expression profiling showed that CXXC finger protein 4 (CXXC4) expression is lower in MCF-7/TMR cells than in MCF-7 cells. Furthermore, CXXC4 mRNA and protein expression are lower in the resistant cell lines than in the corresponding parental cell lines. We also investigated the correlation between CXXC4 and endocrine resistance in ER + breast cancer cells. CXXC4 knockdown accelerates cell proliferation in vitro and in vivo and renders breast cancer cells insensitive to tamoxifen, whereas CXXC4 overexpression inhibits cancer cell growth and increases tamoxifen sensitivity of resistant cells. In addition, we demonstrated that CXXC4 inhibits Wnt/β-catenin signaling in cancer cells by modulating the phosphorylation of GSK-3β, influencing the integrity of the β-catenin degradation complex. Silencing the CXXC4 gene upregulates expression of cyclinD1 and c-myc (the downstream targets of Wnt signaling) and promotes cell cycle progression. Conversely, ectopic expression of CXXC4 downregulates the expression of these proteins and arrests the cell cycle in the G0/G1 phase. Finally, the small-molecule inhibitor XAV939 suppresses Wnt signaling and sensitizes resistant cells to tamoxifen. These results indicate that components of Wnt pathway that are early in response to tamoxifen could be involved as an intrinsic factor of the transition to endocrine resistance, and inhibition of Wnt signaling may be an effective therapeutic strategy to overcome tamoxifen resistance.