Increased G1 Phase Research Articles

The HDAC inhibitor Givinostat modulates the hematopoietic transcription factors NFE2 and C-MYB in JAK2V617F myeloproliferative neoplasm cells

We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)(V617F) myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2(V617F) MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2(V617F) cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2(V617F) cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34(+) cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription.

Abstract 5300: Endothelial colony forming cells (ECFCs) in multiple myeloma

Abstract Introduction: Endothelial progenitor cells (EPCs) have been linked to angiogenesis in many cancers including multiple myeloma (MM). Most published reports on EPCs are based upon myeloid lineage cells, which can support angiogenesis, but cannot form blood vessels on their own. A recently described subpopulation of EPCs called endothelial colony forming cells (ECFCs) have a high clonogenic potential, an ability to differentiate into more mature endothelial cells, as well as in vitro and in vivo vasculogenesis. The role of ECFCs in the pathobiology of MM is unknown. Objective: Determine the in vitro interaction of ECFCs and MM cells by using a matrigel assay, an MTS proliferation assay, and cell cycle analysis. Methods: ECFCs derived from cord blood, or peripheral blood, of normal volunteers and MM patients were combined with human MM cells. MM cells and ECFCs were cultured alone or in co-culture and studied for in vitro tubulogenesis and were evaluated using a 2-dimensional matrigel assay, with images captured at 6 and 24 hours post culture, and analyzed using Image J. Vascular tubules regressed after 24 hours of culture. Therefore, to evaluate the interaction of ECFCs and MM cells over a longer duration, GFP-expressed ECFCs and cherry red expressing MM cells were cultured either alone or in co-culture for 3-5 days. T-tests were used to evaluate the differences between the 2 groups. Results: In matrigel cultures, MM cells preferentially associated with ECFCs as over 90% of MM cells migrated towards the newly formed vascular tubes. No statistical differences in the number of closed units, vascular area and branch length was observed between ECFCs cultured alone or with MM cells. There was also no detectable difference in the proliferation of ECFCs or MM cells. In longer term cultures (3-5 days), the growth of MM cells was significantly reduced in the MM-ECFC co-culture compared to MM cells cultured alone (1.7 Vs. 2.8 fold of baseline, p=0.008). ECFC growth was largely unaffected by MM cells. Summary: Cell cycle analysis showed an increased G1 phase and decreased S phase when MM cells were co-cultured with ECFCs compared to MM cells cultured alone. Conclusion: We demonstrated a tropism of MM cells for ECFCs. In contrast to the proliferation reported when HUVECs are co-cultured with MM cells, ECFCs keep MM cells in a quiescent state by causing G1 arrest. We hypothesize that ECFCs may induce a vascular niche for MM cells. The quiescent state could theoretically result in resistance to chemotherapeutics, which tend to target dividing cells. MM cells could subsequently acquire other molecular events allowing them to dislodge from the vascular niche and contribute to relapse. Additional work to understand the mechanisms underlying the interactions of ECFCs and MM cells, and whether this interaction could be targeted for therapy, are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5300. doi:1538-7445.AM2012-5300

Troglitazone and tumor inhibition: an evolving concept in the management of systemic malignancies

The recent article by An et al. [1] provided for highly stimulating reading. Interestingly, recent data suggests that troglitazone may attenuate tumor growth in a number of systemic malignancies besides accentuating the radio-sensitivity of cervical carcinomas. For instance, troglitazone attenuates tumor growth in gastric malignancies. It mediates this role by modulating the early growth response protein 1 (EGR-1) pathway. It accentuates nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) expression within the cancerous cells [2]. As a result it augments intra-tumoral apoptosis within the gastric carcinomas. These effects are time dependent. Similar effects have been seen in colon carcinomas; it mediates this role by accentuating nuclear factor kappa B (NFκB) inactivation via attenuation of glycogen synthase kinase (GSK)-3β activity within the tumor cells. Intra-tumoral Bax levels are accentuated. As a result apoptosis is markedly augmented [3]. Cyclin B1 and cyclin D1 levels are attenuated. G0/G1 phase arrest is typically seen. Caspase-9 levels are typically accentuated. Troglitazone also decreases FLIP activity and thereby increase the sensitivity of the colon cancer cells to TRAIL induced apoptosis [4]. The anti-neoplastic activity of troglitazone is augmented by loss of X-linked inhibitor of apoptosis protein (XIAP) [5]. Similarly, troglitazone decreases tumor growth in breast cancers. It mediates this role by attenuating human telomerase reverse transcriptase (hTERT) expression within the mammary malignancies [6]. Telomerase activity is markedly reduced. Cdk2 and Cdk4 levels are markedly attenuated. As a result, increased G1 phase arrest is typically seen. In addition, it inhibits histone deacetylase resulting in attenuated phosphatidylinositol 3-kinase (PI3K) signaling [7]. p27 levels are accentuated [8]. These effects are dose dependent. Similar effects are seen in prostate malignancies. Troglitazone primarily exerts these anti-neoplastic effects by accentuating intra-tumoral inactivation of NFκB. It mediates this role by suppression of GSK-3β expression [9]. Troglitazone also mediates this role by augmenting Erk phosphorylation within the cancerous cells. It also modulates p21 and c-myc expression [10]. It down-regulates expression of c-myc. As a result there is increased G0/G1 phase arrest [11]. These effects have been seen both in vivo and in vitro. The above examples clearly illustrate the significant anti-neoplastic activity of troglitazone and the need for further studies in this regard.

Thy28 partially prevents apoptosis induction following engagement of membrane immunoglobulin in WEHI-231 B lymphoma cells

Thy28 protein is conserved among plants, bacteria, and mammalian cells. Nuclear Thy28 protein is substantially expressed in testis, liver, and immune cells such as lymphocytes. Lymphocyte apoptosis plays a crucial role in homeostasis and formation of a diverse lymphocyte repertoire. In this study, we examined whether Thy28 affects induction of apoptosis in WEHI-231 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Once they were established, the Thy28-overexpressing WEHI-231 cells showed similar expression levels of IgM and class I major histocompatibility complex (MHC) molecule compared with controls. The Thy28-overexpressing cells were considerably resistant to loss of mitochondrial membrane potential (ΔΨm), caspase-3 activation, and increase in annexin-positive cells upon mIg engagement. These changes were concomitant with an increase in G1 phase associated with upregulation of p27Kip1. The anti-IgM-induced sustained activation of c-Jun N-terminal kinase (JNK), which was associated with late-phase hydrogen peroxide (H2O2) production, was partially reduced in the Thy28-expressing cells relative to controls. Taken together, the data suggest that in WEHI-231 B lymphoma cells, Thy28 regulates mIg-mediated apoptotic events through the JNK-H2O2 activation pathway, concomitant with an accumulation of cells in G1 phase associated with upregulation of p27Kip1 in WEHI-231 B lymphoma cells.

The role of the tumor suppressor RUNX3 in giant cell tumor of the bone

RUNX3 is a tumor suppressor gene localized in 1p36. In various human tumors, the region is frequently inactivated through hypermethylation, histone modulation and other processes. Recent studies have suggested that loss of RUNX3 expression is involved in stomach, colon and breast cancer. However, the relationship between RUNX3 expression and giant cell tumor of the bone (GCTB) remains elusive. The aim of our study was to elucidate the roles of RUNX3 expression in carcinogenesis and progression of giant cell tumor of the bone. The levels of RUNX3 mRNA and protein were evaluated in human GCTB specimens and cell lines. To assess RUNX3 methylation we employed methylation-specific polymerase chain reaction using GCTB specimens and cell lines. In addition, to examine the roles of RUNX3 in giant cell tumor of the bone, GCTB cells were transfected with pcDNA3.1-RUNX3 (RUNX3 was cloned into the pcDNA3.1 plasmid). Flow cytometry (FCM) was used to analyze the apoptosis and cell cycle. The mobility of cells was tested by transwell migration assay. The expression rates of RUNX3 in patients with GCTB were significanly lower than normal bone tissues. Thirty of 47 human cancer specimens exhibited suppression (P<0.05). Down-regulation of RUNX3 mRNA in the same GCTB cell lines was associated with RUNX3 DNA methylation. In invitro experiments, exogenous expression of RUNX3 strongly inhibited cell growth in GCTB by MTT (P<0.05), induced apoptosis as evidenced by AnnexinV-FITC and increased G1 phase ratio by PI (P<0.05). Transwell migration assay showed that less RUNX3 positive cells migrated to the lower side of the membrane than negative ones (P<0.05). These results show that RUNX3 is a tumor suppressor in GCTB. RUNX3 DNA methylation may be the molecular basis for its lower expression. These data may be applied in GCTB for diagnostics and therapeutics.

Down‐regulation of BMI‐1 cooperates with artemisinin on growth inhibition of nasopharyngeal carcinoma cells

Artemisinin and its derivatives are well known antimalaria drugs, particularly useful for the treatment of infection of Plasmodium falciparum malaria parasites resistant to traditional antimalarial pharmaceuticals. Artemisinin has inhibitory effects on cancer cell growth and anti-angiogenetic activity, including many drug- and radiation-resistant cancer cell lines. Moloney murine leukemia virus insertion site 1 (BMI-1) has been shown to regulate proliferation by inhibiting p16(ink4a) transcription. It is well known that BMI-1 over-expression was found in nasopharyngeal carcinoma cell lines and correlated with advanced invasive stage of the tumor progression and poor prognosis. In the present investigation, we analyzed the inhibitory effects of artemisinin on proliferation of nasopharyngeal carcinoma cell lines (CNE-1 and CNE-2, well-differentiated cells, and poorly differentiated cells). We demonstrated that artemisinin induced G1 cell cycle arrest in CNE-1 and CNE-2 cells. Artemisinin inhibited BMI-1 both in protein and transcript levels. BMI-1 knockdown made the cells more sensitive to artemisinin with an increase in G1 phase, but over-expression of BMI-1 partially reversed the artemisinin-induced G1 cell cycle arrest. Depletion of BMI-1 was able to intensifying the increment of p16 and the reduction of CDK4 induced by artemisinin. In addition, over-expression of BMI-1 was capable of attenuating the increasing p16 and decreasing CDK4 in cells treated with artemisinin. Taking together, the BMI1-p16/CDK4 axis was involved in the artemisinin-driven G1 arrest in nasopharyngeal carcinoma cells, and these results indicated that a potential treatment that the combination of artemisinin and BMI-1 downregulation could enhance the growth inhibitory affects on nasopharyngeal carcinoma cells.

Oncogenic Functions of Secreted Frizzled-Related Protein 2 in Human Renal Cancer

The secreted Frizzled-related proteins (sFRP) are modulators of the Wnt signaling pathway, which is involved in embryonic development and tumor progression. The functions of sFRP2 have not been studied in renal cancer. Transient transfection of sFRP2 promoted cell growth in renal carcinoma cells, whereby the largest effect was observed in A498 cells. To further study the functions of sFRP2 gene in renal carcinoma cells, we established A498 renal cancer cell lines, which stably expressed sFRP2. Stably expressed sFRP2 significantly promoted cell proliferation in vitro and in vivo tumor growth. The stably expressed sFRP2 cells were also found to have reduced UV-induced apoptosis and increased G(2) phase of the cell cycle. The phosphorylation level at Ser(33/37)/Thr(41) of beta-catenin was lower in the stable sFRP2 cell lines compared with the control cell line. sFRP2 significantly activated T-cell factor/lymphoid enhancer factor transcriptional activity. In the stable sFRP2 cell line, expression of c-Fos, Bcl2, Bcl-w, cyclin B2, and cyclin E2 genes was significantly increased and p53 expression was decreased. This is the first report documenting that sFRP2 activates the canonical Wnt pathway and promotes cell growth by evoking diverse signaling cascades in renal cancer cells. This study may provide better strategies for the management of renal cancer through regulation of sFRP2 pathways.

Abstract 4882: Loss of p300 reduces miR-let-7c and enhances ER-positive breast cancer cell tumorigenicity

Abstract Histone deacetylase (HDAC) inhibitors have been extensively studied as potential anticancer treatments and several are currently in clinical trials. p300 is a nuclear histone acetyltransferase that is an important coordinator of multiple transcription-dependent cellular processes. HDAC inhibitors may directly and indirectly increase the activity of p300. The role of p300 is dependent on cell context but most or all of its biological activity stems from its ability to acetylate histones or non-histone nucleoproteins including p53, HSP) which regulate cell proliferation, differentiation and apoptosis. Whether p300 acts as a tumor suppressor or promoter is controversial, and its role in estrogen positive breast cancer remains to be defined, however, structural studies indicate that it has significant potential as a target for small molecule therapy. To address this issue, we have used a lentiviral approach to achieve 60-70% stable knockdown (KD) of p300 levels in estrogen receptor-positive breast cancer lines MCF-7 and BT 474 respectively. Growth curve and FACS analysis showed that loss of p300 shortened doubling times and increased G2 phase cells in both lines. Xenograft tumors formed from p300 KD cells were on average about three times larger at six weeks post implantation than control tumors (p&amp;lt;0.05). H&amp;E and CD31 staining showed that p300 KD xenograft tumors have a more aggressive phenotype with increased vascularity. In searching for targets of p300 that might regulate its anti-tumor effect, we observed that the microRNA miR-let-7c was downregulated in p300-depleted MCF-7 and BT474 cells. Consistent with this, the miR-let-7c target N-Ras was up-regulated in both p300 KD cell lines and in p300 KD xenograft tumors, and adenovirus-mediated over-expression of p300 caused downregulation of N-RAS. Finally, gene expression analysis showed that loss of p300 resulted in upregulation of E-cadherin, laminin, collagen, IL-6, IFN, TGF-β and components of the Wnt signaling pathway, indicating a shift from epithelial to mesenchymal phenotype. Our data show that p300 loss or inactivation results in altered transcription patterns and suppression of anti-tumor microRNAs, including let-7, and may induce a more invasive and potentially metastatic phenotype. We conclude that p300 likely acts as a tumor suppressor in breast cancer and that activation of p300 may contribute to the anti-tumor effects of TSA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4882.

Overexpression of ΔNp63 in a human nasopharyngeal carcinoma cell line downregulates CKIs and enhances cell proliferation

p63 belongs to a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, two types of p63 proteins are produced. The DeltaNp63 isoform lacks the N-terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. DeltaNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of DeltaNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a DeltaNp63 overexpressing human NPC cell line (NPC-076) to explore the possible roles of DeltaNp63 in cell proliferation and cell-cycle regulation. We found that the proliferation of NPC-076 cell is greatly suppressed when the overexpressed DeltaNp63 is silenced by specific DeltaNp63 siRNA. Further studies show that DeltaNp63 silencing results in the upregulation of CKIs, including p27(kip1) and p57(kip2) in both mRNA and protein levels. Cell-cycle analysis shows that DeltaNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that DeltaNp63 plays important roles in the regulation of NPC-076 cell-cycle progression, and may play a role in the maintenance of NPC-076 tumor cell phenotype.

Effects of galectin-3 inhibition on endometrial cell cycle and adhesion

Galectin-3 (gal-3) and its ligands have been implicated in cell transformation and cancer metastasis. Gal-3 protein has been found in uterine epithelial cells adjacent to implanting blastocysts in different cell types. In order to investigate the role of gal-3 in the establishment of human endometrial receptivity, the expression of gal-3 in human endometrial cell line RL95-2 was silenced by RNA interference technology using gal-3 specific small RNA. The expression of gal-3 was detected by the reverse transcriptase-polymerase chain reaction and Western blot analysis. After the suppression of gal-3, cell cycle changes and the expression of integrin β1 were detected by flow cytometry. The adhesive ability of RL95-2 cells was analyzed by the adhesion test. Gal-3 siRNA transfection efficiency reached 70%-90%. The expression of gal-3 mRNA and protein in RL95-2 cells was strongly inhibited by 70%-90% after RNA interference. Inhibition of gal-3 expression decreased S-phase but increased G1 phase cells. Integrin β1 expression was down-regulated, and the adhesive ability of RL95-2 cells to fibronectin (FN) was significantly reduced. Gal-3 may be involved in the establishment of endometrial receptivity by regulating the proliferation and adhesion of endometrial cells. The influence on adhesion may be related with the integrin modulation.

Pyrogallol, ROS generator inhibits As4.1 juxtaglomerular cells via cell cycle arrest of G2 phase and apoptosis

Pyrogallol as a catechin compound has been employed as an O 2 − generator and often used to investigate the role of ROS in the biological system. Here, we investigated the in vitro effect of pyrogallol on cell growth, cell cycle and apoptosis in As4.1 juxtaglomerular cells. Dose-dependent inhibition of cell growth was observed with IC 50 of about 60 μM for 48 h using MTT assay. Pyrogallol (100 μM) did not alter intracellular H 2O 2 level and catalase activity, but increased the intracellular O 2 − level and decreased SOD activity in As4.1 cells. DNA flow cytometric analysis indicated that 50 and 100 μM pyrogallol significantly increased G2 phase cells as compared with those of pyrogallol-untreated cells. Also, pyrogallol induced apoptosis as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay and DAPI staining. This apoptosis process was accompanied with the loss of mitochondrial transmembrane potential (Δ Ψ m), Bcl-2 decrease, caspase-3 activation and PARP cleavage. Pan caspase inhibitor (Z-VAD) could significantly rescue As4.1 cells from pyrogallol-induced cell death. But, the inhibitors of caspase-3, caspase-8, and caspase-9 did not prevent apoptotic events in pyrogallol-treated As4.1 cells. Taken together, we have demonstrated that an ROS inducer, pyrogallol inhibits the growth of As4.1 JG cells via cell cycle arrest and apoptosis, and suggest that the compound exhibits an anti-proliferative efficacy on these cells.

Phosphorylation of HuR by Chk2 Regulates SIRT1 Expression

The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.

IKKα controls p52/RelB at the skp2 gene promoter to regulate G1- to S-phase progression

The IkappaB-inducing kinase (IKK) is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma. IKK-regulated signaling pathways are believed to promote the proliferation of normal cells as well as the aberrant proliferation of cancer cells. The molecular mechanisms linking the IKK signaling pathway components to the cell cycle machinery are not entirely understood. To study the function(s) of the catalytic subunits of the IKK complex, we used pancreatic cancer cells, with constitutive IKK activity. We show that the G1 phase of the cell cycle is specifically regulated by the IKKalpha subunit, which regulates the stability of the cyclin-dependent kinase inhibitor p27(Kip1). Increased p27(Kip1) protein levels following the transfection of IKKalpha-specific siRNAs are a result of the downregulation of the F-box protein S-phase kinase-associated protein 2 (skp2). Additionally, we demonstrate that IKKalpha signaling regulates the transcription of the skp2 gene by controlling the composition of a RelB-containing NF-kappaB complex. Together, this work defines a novel IKKalpha-regulated growth pathway involving the p52/RelB-dependent transcriptional regulation of the skp2 gene.

STI‐571 (Gleevec) Potentiates the Effect of Cisplatin in Inhibiting the Proliferation of Head and Neck Squamous Cell Carcinoma In Vitro

The objective of this study was to determine whether STI-571 (Gleevec; imatinib mesylate) could sensitize established head and neck squamous cell carcinoma (HNSCC) cell lines to the effects of cisplatin. Western blot analysis and immunofluorescence were used to examine the expression of the tyrosine kinases that are known targets of Gleevec, including c-kit, c-Abl, and platelet-derived growth factor receptor, on the cell lines, and immunohistochemistry was performed to determine the expression of these kinases in human HNSCC tissue. Once these targets were confirmed, clonogenic cell survival assays were performed to determine the effect STI-571 had on growth and proliferation when used in combination with cisplatin compared with STI-571 alone or cisplatin alone. Cells were incubated with a range of doses of STI-571 24 hours before the addition of cisplatin. Flow cytometry analysis was performed to determine cell-cycle distribution and to measure apoptosis caused by the various treatments. An annexin V assay was also used to further measure apoptosis. Our results indicate that STI-571 potentiates the effect of cisplatin and leads to a significant decrease in cell proliferation and colony formation compared with cisplatin alone in a dose-dependent fashion. Surprisingly, there was a slight decrease in the level of apoptosis when Gleevec was used in combination with cisplatin compared with cisplatin alone. Gleevec alone resulted in a slight increase in G1 phase of the cell cycle, whereas cisplatin alone resulted in a G2 arrest. The addition of Gleevec to cisplatin resulted in an enhanced S/G2 phase accumulation. Although we did not demonstrate an increase in cisplatin-induced cell death, we postulate that the increased S/G2 arrest resulting from the DNA damage in the presence of Gleevec results in decreased proliferation of HNSCC, resulting in a net decrease in colony formation. The small molecule inhibitor Gleevec, which targets specific tyrosine kinases that are expressed in HNSCC, can significantly potentiate the antiproliferative effects of cisplatin. Because Gleevec alone has minimal side effects, treatment with the combination treatment of cisplatin and Gleevec may result in increased efficacy of cisplatin in treating this cancer. Additional studies are warranted, keeping in mind that drug combinations may result in unexpected toxicities that are not frequently seen with either drug alone.

하인두암 세포주에서 Cisplatin, 5-Fluorouracil, 방사선조사가 세포주기 및 세포고사에 미치는 영향

Objectives and Background:In head and neck cancer including hypopharyngeal cancer, cisplatin and 5- fluorouracil (5-FU) usually have been used as neoadjuvant chemotherapeutic agents. We investigated the difference of the influences of cisplatin, 5-FU and radiation on p53 protein expression and cell responses (cell cycle arrest and/or apoptosis) in the hypopharyngeal carcinoma cell line (PNUH-12 ; mutant-type p53). Method:PNUH-12 cells were treated with cisplatin, 5-FU and radiation. The changes in the cells were assessed by acell cytotoxicity assay, Western blotting (p53 and p21WAFI/CIPI proteins), DNA fragmentation assay, pro- pidium iodide (PI) stain and DNA flow cytometry. Results:The expression of p53 protein was increased after treatment with cisplatin and 5-FU, but not radiation. The expression of p21WAF1/CIPI protein was increased only after treatment with 5-FU, not cisplatin or radiation. With cisplatin and radiation, we observed apoptosis in both by DNA fragmentation and PI stain and increased the S phase in cisplatin and the G2 phase in radiation by DNA flow cytometry. But, with 5-FU, we couldn’t observe apoptosis by DNA fragmentation and PI stain but only an increased G1 phase by DNA flow cytometry. Conclustions:In PNUH-12, radiation induced p53- independent apoptosis and p21WAFI/CIPI-mdependent G2 phase cell-cycle arrest. Cisplatin induced p53-depen- dent apoptosis and p21WAF1/C1P1-independent S phase cell-cycle arrest and 5-FU induced p53 and p21WAF1/CIPI- dependent G1 phase cell-cycle arrest, not apoptosis. Cisplatin and 5-FU induced p53-dependent pathways, but radiation p53-independent pathway. The cell responses by cisplatin, 5-FU and radiation were all different pathways. Our results suggest that combined treatmentwith radiation and cisplatin or 5-FU may be effect because these have different molecular mechanism of cell damage. (J Clinical Otolaryngol 2005;16:45-53)

Inhibitory effect of p27KIP1 gene transfer on head and neck squamous cell carcinoma cell lines.

Although p27 gene mutations are rarely found in cancer, the level of p27 protein expression decreases during tumor development. In several tumors, including laryngeal cancer, decreased expression of p27 is associated with a poor prognosis. The proliferation-inhibitory effect of p27 gene transfer on human head and neck squamous cell carcinoma (HNSCC) cell lines (SNU-1041, SNU-1066, and SNU-1076) by adenoviral vector was investigated. On transduction of the human HNSCC cell line with adenovirus-p27 (Ad-p27), a high level of p27 expression and increase of cyclin D1 and E were observed. Cell cycle analysis showed a marked decrease in the proportion of cells in S phase and an increase in G1 phase. Soft agar clonogenic assay showed a marked decrease in clonogenicity. These results suggested that overexpression of p27 could show proliferation-inhibitory effects on HNSCC cell lines. Thus, gene therapy using adenovirus-p27 seemed to have a potential to develop into a new cancer gene therapy modality.

Effects of Adenovirus-mediated Expression of p27 Kip1, p21 Waf1 And p16 INK4A in Cell Lines Derived from t(2;5) Anaplastic Large Cell Lymphoma and Hodgkin's Disease

We investigated the response of SUDHL-1 and L428 cells, derived from t(2;5)-anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD), respectively, to recombinant adenoviruses expressing cyclin-dependent kinase inhibitors (CDKIs) p27 Kip1 (Adp27), p21 Waf1 (Adp21) and p16 INK4A (Adp16). Cell cycle analysis of SUDHL-1 cells after 24 h of infection with 200 multiplicity of infection (MOI) of Adp27, Adp21, and Adp16, showed very high levels of cell debris in the subG1 area. The magnitude of cell debris-events was Adp27/Adp21>Adp16. Cell cycle analysis of L428 cells revealed absence of cell debris and increased G2 phase in all the groups of cells tested as compared to the controls (mock and AdNull). A minimal increase in G1 phase was also evident in cells infected with Adp27 (52%) compared to uninfected cells (43%), AdNull (45%) and to cells infected with Adp21 (37%) and Adp16 (31%). The presence of significant levels of Coxsackie-adenovirus receptor (CAR) on the cell surface of L428 cells excluded the cell membrane-barrier as responsible for the differences in cell observed in response to the recombinant adenovirus-mediated CDKIs expression as compared to SUDHL-1. We also showed that the recombinant adenovirus-mediated cytotoxicity measured as apoptosis was MOI- and vector-dependent in SUDHL-1 cells at lower MOI (100). In conclusion, the therapeutic effect induced by recombinant adenoviruses expressing p27 Kip1, p21 Waf1 and p16 INK4A is cell-dependent in cells derived from selected lymphoid malignancies. Biochemical cellular differences more than cell surface barriers seem to be responsible for differences in response to recombinant adenovirus-mediated expression of cytotoxic genes. Moreover, the cytotoxicity of recombinant adenoviruses expressing p27 Kip1, p21 Waf1 and p16 INK4A may be further explored as a tool for gene therapy of t(2;5)-derived ALCL.

Role of inducible heat shock protein 70 in radiation-induced cell death.

We previously demonstrated the protective effect of inducible heat shock protein 70 (Hsp70) against gamma radiation. Herein, we extend our studies on the possible role of Hsp70 to ionizing radiation-induced cell cycle regulation. The growth rate of inducible hsp70-transfected cells was 2-3 hours slower than that of control cells. Flow cytometric analysis of cells at G1 phase synchronized by serum starvation also showed the growth delay in the Hsp70-overexpressing cells. In addition, reduced cyclin D1 and Cdc2 levels and increased dephosphorylated phosphoretinoblastoma (pRb) were observed in inducible hsp70-transfected cells, which were probably responsible for the reduction of cell growth. To find out if inducible Hsp70-mediated growth delay affected radiation-induced cell cycle regulation, flow cytometric and molecular analyses of cell cycle regulatory proteins and their kinase were performed. The radiation-induced G2/M arrest was found to be inhibited by Hsp70 overexpression and reduced p21Waf induction and its kinase activity by radiation in the Hsp70-transfected cells. In addition, radiation-induced cyclin A or B1 expressions together with their kinase activities were also inhibited by inducible Hsp70, which represented reduced mitotic cell death. Indeed, hsp70 transfectants showed less induction of radiation-induced apoptosis. When treated with nocodazole, radiation-induced mitotic arrest was inhibited by inducible Hsp70. These results strongly suggested that inducible Hsp70 modified growth delay (increased G1 phase) and reduced G2/M phase arrest, subsequently resulting in inhibition of radiation-induced cell death.

Partial synchronization of cell division in cultured maize (Zea mays L.) cells: differential cyclin, cdc2, histone, and retinoblastoma transcript accumulation during the cell cycle

Optimization of culture and treatment conditions for reproducible synchronization of the cell cycle in maize cells with hydroxyurea is described. Flow cytometric measurements of relative DNA contents revealed that, following the hydroxyurea block, around 55% of the cells were in the G1 phase (2C), 30% in the S phase (2C 4C) and 15% in the G2 phase (4C) of the cell cycle. The highest frequencies of cells in the S phase could be observed 2 h after removal of hydroxyurea (35-40%). The cells reached increased G2 phase frequencies (60-70%) between 8-14 h, while the maximum number of mitotic cells (12-14%) were found between 14-17 h. Northern analysis of total RNA from the synchronized cells indicated an increased level of transcripts from two histone (H3Zm, H4Zm) genes during the S-phase. The changes in the mRNA levels of the maize cyclin variants CycB1;zm;1, CycB1;zm;2 and CycA1;zm;1 support the classification of these cyclins as mitotic cyclins with transcript accumulation during the G 2 /M cell cycle phases. Using the maize retinoblastoma (ZmRb-1) cDNA as hybridization probe, two transcripts were detected with different hybridization intensity: the smaller, more abundant transcript was recognized during the whole cell cycle with an increase after the release of the cells from the hydroxyurea block, while the larger mRNA could only be detected for 8h after removal of hydroxyurea. The ZmRb-1 gene might respond to the increase of the frequency of cells in the S phase or to the re-addition of conditioned medium to the cells after hydroxyurea removal.

Simultaneous measurement, using flow cytometry, of radiosensitivity and defective mitogen response in ataxia telangiectasia and related syndromes.

In a retrospective study, peripheral blood mononuclear cells from 13 patients with known ataxia telangiectasia (AT) (Louis Bar syndrome, McKusick #20890) were irradiated with different doses of X-rays prior to stimulation with phytohaemagglutinin. Mitogen response and cell cycle progression were assessed by two-parameter 5-bromo-2'-deoxyuridine/Hoechst--ethidium bromide flow cytometry. Compared to age-matched controls, AT cells show a severely defective mitogen response in both unirradiated and irradiated cells. Following irradiation with 1.5 Gy, AT cells exhibit significantly greater accumulations of cells in the G2 phase of the first cell cycle than controls. The ratio between the number of cells accumulated in the first cycle G2 phase and the growth fraction provides a clear distinction between AT and control cultures. In addition, two patients with microcephaly, normal intelligence, immunodeficiency, chromosomal instability and risk for lymphoreticular malignancies (Seemanová syndrome) and two patients with the Nijmegen breakage syndrome (both syndromes are listed as McKusick #25126) also exhibit very poor mitogen response and moderately increased G2 phase accumulations after X-irradiation. The simultaneous assessment of radiosensitivity and mitogen response in a single cell kinetic assay provides a speedy and accurate classification of cells of AT and AT-related syndromes.