G1-phase Fraction Research Articles

Chk1/2 inhibitor AZD7762 blocks the growth of preantral follicles by inducing apoptosis, suppressing proliferation, and interfering with the cell cycle in granulosa cells.

Checkpoint kinases 1/2 (Chk1/2) have an important role in somatic cell development and oocyte meiotic maturation. However, the role of Chk1/2 in folliculo-genesis has not been fully elucidated. The aim of this study was to assess the effects of Chk1/2 inhibition on ovarian folliculogenesis and granulosa cell development in mice. Preantral follicles (100-120 μm) and granulosa cells from pre-ovulatory follicles (pre-GCs) of mice were isolated and cultured with or without Chk1/2 inhibitor AZD7762. Preantral follicles were cultured for 96h. Then, follicle morphology and fol-licular growth were assessed every 48h. Granulosa cells were cultured for 48h with or without AZD7762, after which cell apoptosis, cell proliferation, and cell cycle analysis were assessed; meanwhile, the mRNA expression of PCNA and Bax were measured by real-time RT-PCR, and PCNA and Bax protein were measured by Western blot. Compared with control follicles, AZD7762 inhibited growth of preantral folli-cles (P<0.05). Furthermore, inhibition of Chk1/2 significantly induced apoptosis (P<0.05) and inhibited the proliferation of granulosa cells (P<0.01), arrested cell cycle at S and G2/M phases, and decreased G1 phase fraction (P<0.001). Also, the expres-sion of PCNA mRNA and protein were reduced (P<0.01), while Bax mRNA and pro-tein were increased (P<0.05) post AZD7762 treatment in granulosa cells. This study revealed that Chk1 and Chk2 have a crucial role during preantral follicular development by regulating the proliferation and apoptosis of granu-losa cells.

Anti-tumour effect of lapatinib in canine transitional cell carcinoma cell lines.

Transitional cell carcinoma (TCC) accounts for >90% of canine malignant tumours occurring in urinary bladder, and the prognosis is poor. Our previous study, using RNA sequencing, showed that human epidermal growth factor 2 (HER2) was the most activated upstream regulator related to carcinogenesis in canine TCC. The aim of this study was to examine the anti-tumour effect of lapatinib, a tyrosine kinase inhibitor of HER2, on canine TCC cell lines in vitro and in vivo. Five canine TCC cell lines (TCCUB, Love, Sora, LCTCC, and MCTCC) were used. Western blotting showed that HER2 protein expression was observed in all of the canine TCC cell lines. Lapatinib inhibited phosphorylation of HER2 and cell growth in a dose-dependent manner. Cell cycle analyses using flow cytometry showed that lapatinib significantly increased the sub-G1 and G0 /G1 phase fractions and significantly decreased the S and G2 /M phase fractions in the cell lines (Sora and TCCUB). For the in vivo experiments, the canine TCC cells (Sora) were subcutaneously injected into nude mice. Six days after inoculation, lapatinib (100 mg/kg) or vehicle was administered daily via intraperitoneal administration for 14 days. Tumour volume was significantly smaller in the lapatinib group compared with the vehicle control group. Histologically, lapatinib significantly increased necrotic areas in the tumour tissues. These findings suggest that lapatinib exerts anti-tumour effects on canine TCC cells by inhibiting HER2 signalling and inducing cell cycle arrest.

The effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer.

Centromere protein U (CENPU) is a novel transcriptional repressor that is associated with different types of cancer. However, its function in breast cancer is poorly understood. In the present study, it was identified that CENPU was highly expressed in breast cancer tissues compared with expression in normal breast tissues (P=0.001). Furthermore, the CENPU mRNA level in tumors was often elevated, compared with the matched adjacent normal breast cancer tissue specimens in the dataset from The Cancer Genome Atlas database (n=106; P<0.001). To understand the function of CENPU in human breast carcinogenesis, its effects on the proliferation, apoptosis and cell cycle progression of MDA-MB-231 cells were examined using the lentiviral-mediated CENPU knockdown approach. The RNA and protein expression levels in the transfected cells were monitored using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The mRNA and protein expression levels of the CENPU gene were significantly lower in the CENPU-shRNA transfected cells than in the control (P<0.01), indicating successful gene expression knockdown. Post-transfection, cell counting and MTT analysis revealed that the proliferation activity was significantly suppressed in CENPU knockdown cells relative to the control (P<0.01). Additionally, fluorescence activated cell sorting analysis revealed that the (G2+S) phase fraction was significantly declined in CENPU knockdown cells relative to the control; while the G1 phase fraction was significantly increased (P<0.01) and the percentage of the apoptotic cells was significantly increased (P<0.01). In conclusion, downregulation of CENPU gene expression may inhibit cell proliferation and cell cycle progression, and increase the apoptosis of the breast cancer cells. These results suggested a possible function of this protein in breast cancer pathogenesis and prognosis.

Insights Into SMAD4 Loss in Pancreatic Cancer From Inducible Restoration of TGF-β Signaling.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer death in the United States. The TGF-β signaling protein SMAD family member 4 is lost in 60% of PDAC, and this has been associated with poorer prognosis. However, the mechanisms by which SMAD4 loss promotes PDAC development are not fully understood. We expressed SMAD4 in human PDAC cell lines BxPC3 and CFPAC1 by selection of stable clones containing an inducible SMAD4 tetracycline inducible expression system construct. After 24 hours of SMAD4 expression, TGF-β signaling-dependent G1 arrest was observed in BxPC3 cells with an increase in the G1 phase fraction from 48.9% to 71.5%. Inhibition of cyclin-dependent kinase inhibitor 1A by small interfering RNA eliminated the antiproliferative effect, indicating that up-regulation of cyclin-dependent kinase inhibitor 1A/p21 by TGF-β signaling is necessary for the phenotype. SMAD4 expression had no impact on invasion in BxPC3 cells, but reduced migration. Microarray analysis of gene expression at 8, 24, and 48 hours after SMAD4 expression characterized the regulatory impact of SMAD4 expression in a SMAD4-null PDAC cell line and identified novel targets of TGF-β signaling. Among the novel TGF-β targets identified are anthrax toxin receptor 2 (3.58× at 8 h), tubulin, β-3 class III (7.35× at 8 h), cell migration inducing protein, hyaluronan binding (8.07× at 8 h), IL-1 receptor-like 1 (0.403× at 8 h), regulator of G protein signaling 4 (0.293× at 8 h), and THAP domain containing 11 (0.262× at 8 h). The gene expression changes we observed upon restoration of TGF-β signaling provide numerous new targets for future investigations into PDAC biology and progression.

Bioinspired Phospholipid Polymer Hydrogel System for Cellular Engineering

SummaryThe properties of the microenvironment surrounding cells are important for the control of cell functions. The polymeric cellular environment has great potential in this regard because environmental properties can be manipulated. We propose a spontaneously forming phospholipid polymer hydrogel system composed of 2 kinds of pre‐polymers to control cellular functions via hydrogel physical properties. These pre‐polymers are cytocompatible poly(2‐methacryloyloxyethyl phosphorylcholine‐co‐n‐butyl methacrylate‐co‐p‐vinylphenylboronic acid) (PMBV) and water‐soluble poly(vinyl alcohol) (PVA). The p‐vinylphenylboronic acid units in PMBV can react with the hydroxy groups of PVA in aqueous medium and form cross‐linkages. This spontaneously formed PMBV/PVA hydrogel can be dissociated again via the addition of sugar compounds. To alter the physical properties, we simply change the concentration or mixing ratio of the pre‐polymers. The storage modulus of the PMBV/PVA hydrogel matrix was controlled from 0.3 kPa to 2.5 kPa, which corresponds to very soft natural tissue. Cells can be encapsulated in the hydrogel. When the storage modulus of the PMBV/PVA hydrogel was above 1.0 kPa, the proliferation of encapsulated cells was suppressed and provided uniform cells in G1 phase in cell cycle progression. High G1 phase fraction (&gt;90%) may lead to excellent differentiation efficiency, which results in great insight for stem cell engineering. The PMBV/PVA system is expected to become a key material in cellular engineering for regulating cellular function without undesirable biological events.

The value of cell cycle analysis by propidium-iodine staining of CD56+ cells in pediatric brain tumors

PurposeCell cycle analysis by flow cytometry has not been adequately studied in pediatric brain tumors. We investigated the value of cell cycle analysis by propidium-iodine (PI) staining of CD56+ (gated) cells for the characterization of pediatric brain tumor's malignancy. MethodsPatients that underwent surgery for an intracranial lesion and tissue sample was available were included in the study. All tumor samples were histopathologically verified before flow cytometric analysis. ResultsThere were 46 pediatric brain tumor cases. As control we used samples from three cases of brain tissue obtained during surgery for epilepsy. All tumors had significant lower G0/G1 and significant higher S-phase, G2/M fraction, S+G2/M and S+G2/M/G0/G1-phase fraction than normal brain tissue. Low-grade tumors had significant lower S-phase than high grade tumors. Grade IV tumors had significant lower G0/G1 fraction and higher S+G2/M/G0/G1 index than grade III tumors. DNA diploidy was detected in 24 tumors and DNA aneuploidy was detected in 23 tumors. There was a significant correlation between Ki-67 index and both S+G2/M and S+G2/M/G0/G1 phase fraction. ConclusionsCell cycle analysis by PI staining of CD56+ cells is a promising method for the assessment of pediatric brain tumors malignancy and could be a valuable adjunct to histopathological evaluation.

Caspase-mediated pro-apoptotic interaction of panaxadiol and irinotecan in human colorectal cancer cells

Panaxadiol is a purified sapogenin of ginseng saponins that exhibits anticancer activity. Irinotecan is a second-line anticancer drug, but clinical treatment with irinotecan is limited due to its side effects. In this study, we have investigated the possible synergistic anticancer effects of panaxadiol and irinotecan on human colorectal cancer cells and explored the potential role of apoptosis in their synergistic activity. The combination of panaxadiol and irinotecan significantly enhanced antiproliferative effects in HCT-116 cells (P< 0.05). Cell cycle analysis demonstrated that combining irinotecan treatment with panaxadiol significantly increased the G1-phase fractions of cells, compared with irinotecan treatment alone. In apoptotic assays, the combination of panaxadiol and irinotecan significantly increased the percentage of apoptotic cells compared with irinotecan alone (P<0.01). Increased activity of caspase-3 and caspase-9 was observed after treating with panaxadiol and irinotecan. The synergistic apoptotic effects were supported by docking analysis, which demonstrated that panaxadiol and irinotecan bound two different chains of the caspase-3 protein. Data from this study suggested that caspase-3- and caspase-9-mediated apoptosis may play an important role in the panaxadiol enhanced antiproliferative effects of irinotecan on human colorectal cancer cells.

Different responses between diploid and tetraploid H1 embryonic stem cells appeared during long-term culturing in L15F10 medium without leukemia inhibitory factor

To examine the alteration in cellular characteristics of polyploid embryonic stem (ES) cells during long-term culturing without leukemia inhibitory factor (LIF), mouse diploid and tetraploid H-1 (ES) cells (2H1 and 4H1 cells, respectively) were cultured without LIF for approximately 5 months. 2H1 and 4H1 cells were adapted to the medium without LIF by decreasing the concentration for several passages, and they were denoted as 2H1(⁻) and 4H1(⁻) cells, respectively. DNA content of 4H1(⁻) cells decreased gradually in the early stage, increased abruptly in the second stage, and then was maintained for a long time. 4H1(⁻) cells exhibited longer doubling time and equivalent phase fraction compared with those of 2H1(⁻) cells. The G₁ phase fractions of 2H1(⁻) and 4H1(⁻) cells were increased compared with that of 2H1 cells. Cellular morphology and pluripotency were maintained in 4H1(⁻) cells but not in 2H1(⁻) cells. 2H1(⁻) cells showed a cell population consisting of several kinds of cells, and they lost alkaline phosphatase activity, suggesting that the cells had differentiated. 4H1(⁻) cells, however, exhibited alkaline phosphatase activity and formed teratocarcinoma in mouse abdomen, suggesting that the cells maintained their pluripotency in the medium without LIF.

Inositol Hexaphosphate (IP6): Modulation of Cell Cycle and Proliferation of Bladder Cancer in vitro

Introduction: We hypothesized that inositol hexaphosphate (IP6) would modulate cell cycle and cellular growth in bladder cancer. Materials and Methods: Bladder cancer cell lines were treated with 2.5 or 4.5 mM/well IP6. Cell viability was measured by MTT at 24 and 48 h. Cell cycle analysis was measured by DNA staining and quantified by FACS analysis. Results: Cell growth was inhibited in the TCCSUP, HTB9 and T24 cells (p < 0.001) with the exception of the HTB9 cells at 24 h. G1 phase fractions increased in the TCCSUP and HTB9 cells, while S phase fraction was decreased (p < 0.001). G2 phase was decreased in the TCCSUP cells (p < 0.001). G1 phase fraction was decreased in the T24 line with 4.5 mMIP6 (p < 0.001), while a significant increase in the S phase was observed in the T24 cells (p < 0.001). Conclusions: These results indicate that IP6 is potentially a highly effective treatment for carcinoma of the bladder.

Cell Proliferation, Cell Cycle Abnormalities, and Cancer Outcome in Patients with Barrett's Esophagus: A Long-term Prospective Study

Elevated cellular proliferation and cell cycle abnormalities, which have been associated with premalignant lesions, may be caused by inactivation of tumor suppressor genes. We measured proliferative and cell cycle fractions of biopsies from a cohort of patients with Barrett's esophagus to better understand the role of proliferation in early neoplastic progression and the association between cell cycle dysregulation and tumor suppressor gene inactivation. Cell proliferative fractions (determined by Ki67/DNA multiparameter flow cytometry) and cell cycle fractions (DNA content flow cytometry) were measured in 853 diploid biopsies from 362 patients with Barrett's esophagus. The inactivation status of CDKN2A and TP53 was assessed in a subset of these biopsies in a cross-sectional study. A prospective study followed 276 of the patients without detectable aneuploidy for an average of 6.3 years with esophageal adenocarcinoma as an end point. Diploid S and 4N (G(2)/tetraploid) fractions were significantly higher in biopsies with TP53 mutation and loss of heterozygosity. CDKN2A inactivation was not associated with higher Ki67-positive, diploid S, G(1), or 4N fractions. High Ki67-positive and G(1)-phase fractions were not associated with the future development of esophageal adenocarcinoma (P=0.13 and P=0.15, respectively), whereas high diploid S-phase and 4N fractions were (P=0.03 and P<0.0001, respectively). High Ki67-positive proliferative fractions were not associated with inactivation of CDKN2A and TP53 or future development of cancer in our cohort of patients with Barrett's esophagus. Biallelic inactivation of TP53 was associated with elevated 4N fractions, which have been associated with the future development of esophageal adenocarcinoma.

Additive effects of Cox-1 and Cox-2 inhibition on breast cancer in vitro

We hypothesized that combined treatment with Cox-1 and Cox-2 specific inhibitiors would exhibit synergistic effects against breast cancer in vitro. Two human breast cancer cell lines (HTB26, MCF-7) were treated with catechin (Cox-1 inhibitor) or NS398 (Cox-2 inhibitor) at 100 microM as both single and combined treatments. Reductions in cell growth were observed in both cell lines at 24 and 72 h in both single and combined treatments (p<0.001). Combined treatment produced a significantly greater inhibition as compared to single agents alone. Upon cell cycle evaluation, Cox-1 and -2 antagonism increased G1 and G2 phase fractions in MCF-7 cells (p<0.001 and p<0.05 respectively). No additive changes were observed when the two agents were combined. An increase in the G2 phase was observed in the HTB26 cells when treated with NS398 alone (p<0.001). However, a decrease in the S-phase was observed when these cells were treated with NS398, as a single agent (p<0.01) or when the two agents were combined (p<0.01). The significant and additive effects exhibited by the combination of Cox-1 and -2 inhibitors and their effects on cell cycle suggest that these agents could become an effective treatment modality for carcinoma of the breast.

Inhibition of AKT/mTOR Signaling Pathway Induces Cell Cycle Arrest and Apoptosis in Acute Myelogenous Leukemia.

Acute myelogeneous leukemia (AML) is a heterogeneous disease and includes a subset of neoplasms that harbor activating mutations of the fms-like tyrosine kinase-3 (FLT3) gene. Mutated FLT3 has recently been shown to activate downstream oncogenic pathways including the PI3K/AKT pathway (Scheijen, et al. Oncogene . 23:3338–3349, 2004; Choudhary, et al. Blood . 106:265–273, 2005). It is known that activated AKT mediates its effects, at least in part, through activation of mammalian target of rapamycin (mTOR). However, the potential role of PI3K/AKT/mTOR signaling pathway in tumor cell survival in AML remains largely unknown. We hypothesized that the PI3K/AKT signaling pathway is activated in AML and contributes to tumor cell survival through activation (phosphorylation) of mTOR and its downstream effectors 4EBP1, p70S6K, ribosomal protein S6 (rpS6), and eIF-4E. We used 3 AML cell lines, including MV4-11 and MOLM-13, that are homozygous and heterozygous for mutated FLT3, respectively, as well as U937 (wild-type FLT3). All 3 cell lines expressed activated (serine 473-phosphorylated) AKT (Ser473pAKT), and phosphorylated 4EBP1, p70S6K and rpS6 shown by Western Blot analysis. Treatment of AML cell lines with LY294002, an inhibitor of PI3K, resulted in a dose-dependent decrease of phosphorylation of AKT, mTOR, 4EBP1, p70S6K, and rpS6. This was associated with decreased cell viability as assessed by trypan-blue exclusion assay. Cell death following inhibition of the PI3K/AKT pathway was predominantly attributed to apoptosis as shown by increased annexin V staining assessed by flow cytometry. These changes were associated with downregulation of the anti-apoptotic proteins cFLIP, Mcl-1, and Bcl-XL that are involved in the extrinsic and intrinsic apoptosis. Cell cycle analysis using flow cytometry also showed that inhibition of PI3K resulted in decreased S-phase and increased G1-phase fraction. These cell cycle changes were associated with increased levels of the cyclin-dependent kinase inhibitor p27 and underphosphorylated Rb in a dose-dependent manner. Similar biologic effects, although to a lesser degree, were found after treatment of AML cells with rapamycin, an inhibitor of mTOR. In addition, expression of activated AKT, mTOR, 4EBP1, p70S6K and rpS6 was assessed in AML tumors (n=19) using tissue microarrays of bone marrow samples and immunohistochemical methods. These included tumors with (n=14) and without (n=5) FLT3 mutations. Using a 10% cutoff to define positivity, 13/19 (68%) expressed Ser473pAKT, 16/18 (89%) mTOR, 15/19 (79%) p4E-BP1, 18/19 (95%) p-p70S6K, and 15/18 (83%) p-rpS6. However, no association between expression of activated AKT, or mTOR signaling proteins and FLT3 mutational status was observed. Our study provides first evidence that the AKT/mTOR signaling pathway is activated in AML cell lines and tumors regardless of FLT3 mutational status. The AKT/mTOR signaling pathway may contribute to cell cycle progression and tumor cell survival in AML. Inhibition of this oncogenic pathway represents a potential target for therapy in patients with AML.

Onychin inhibits proliferation of vascular smooth muscle cells by regulating cell cycle1

To investigate the effects of onychin on the proliferation of cultured rat artery vascular smooth muscle cells (VSMCs) in the presence of 10% new-born calf serum (NCS). Rat VSMCs were incubated with onychin 150 micromol/L or genistein 10 micromol/L in the presence of 10% NCS for 24 h. The proliferation of VSMCs was measured by cell counting and MTS/PMS colorimetric assays. Cell cycle progression was evaluated by flow cytometry. Retinoblastoma (Rb) phosphorylation, and expression of cyclin D1 and cyclin E were measured by Western blot assays. The tyrosine phosphorylation of ERK1/2 was examined by immunoprecipitation techniques using anti-phospho-tyrosine antibodies. The proliferation of VSMCs was accelerated significantly in the presence of 10% NCS. Onychin reduced the metabolic rate of MTS and the cell number of VSMCs in the presence of 10% NCS in a dose-dependent manner. Flow cytometry analysis revealed that the G1-phase fraction ratio in the onychin group was higher than that in the 10% NCS group (85.2% vs 70.0%, P<0.01), while the S-phase fraction ratio in the onychin group was lower than that in 10% NCS group (4.3% vs 16.4%, P<0.01). Western blot analysis showed that onychin inhibited Rb phosphorylation and reduced the expression of cyclin D1 and cyclin E. The effects of onychin on proliferation, the cell cycle and the expression of cyclins in VSMCs were similar to those of genistein, an inhibitor of tyrosine kinase. Furthermore immunoprecipitation studies showed that both onychin and genistein markedly inhibited the tyrosine phosphorylation of ERK1/2 induced by 10% NCS. Onychin inhibits the proliferation of VSMCs through G1 phase cell cycle arrest by decreasing the tyrosine phosphorylation of ERK1/2, and the expression of cyclin D1 and cyclin E, and sequentially inhibiting Rb phosphorylation.

Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells.

Amplification of MYCN is one of the most important prognostic markers for neuroblastoma and is correlated with rapid tumor progression and poor prognosis. MYCN belongs to the Myc/Max/Mad/Mnt network of proteins that regulate proliferation, apoptosis, and differentiation. It is well established that MYCN is downregulated during induced differentiation of neuroblastoma cells carrying an amplified MYCN gene, but very little is known about other components of the network, i.e., the Max, Mad, and Mnt proteins, during this process. In this study we show that Mad and Mnt expression was only modestly regulated in differentiating SK-N-BE(2) neuroblastoma cells, while MYCN was rapidly downregulated. This downregulation was reflected in a decreased MYCN/Max DNA-binding activity while the Mnt/Max binding did not change during differentiation. In parallel experiments we also analyzed the Myc/Max/Mad expression and DNA binding capacity during induced differentiation in the MYCN single copy neuroblastoma cell line SH-SY5Y. In this cell line only modest changes in expression of the components of the MYCN/Max/Mad/Mnt network was detected, but since the cell line expresses relatively low levels of MYCN and c-Myc, these changes might be of functional significance. Cell cycle analyses of SK-N-BE(2) demonstrated an increase in the G1-phase fraction after RA-treatment. These data show that the decreased MYCN expression and MYCN DNA-binding is correlated with retarded cell cycle progression. Furthermore, when Mad1 or Mnt was overexpressed in SK-N-BE(2) cells they retained the capacity to differentiate, underscoring the notion that MYCN downregulation, and not changes in Mad/Mnt expression, is essential for neuroblastoma cell differentiation.

Cell density related gene expression: SV40 large T antigen levels in immortalized astrocyte lines

BackgroundGene expression is affected by population density. Cell density is a potent negative regulator of cell cycle time during exponential growth. Here, we asked whether SV40 large T antigen (Tag) levels, driven by two different promoters, changed in a predictable and regular manner during exponential growth in clonal astrocyte cell lines, immortalized and dependent on Tag.ResultsExpression and cell cycle phase fractions were measured and correlated using flow cytometry. T antigen levels did not change or increased during exponential growth as a function of the G1 fraction and increasing cell density when Tag was transcribed from the Moloney Murine Leukemia virus (MoMuLV) long terminal repeat (LTR). When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased. When transcribed from the herpes thymidine kinase promoter, Tag levels decreased. The directions of change and the rates of change in Tag expression were unrelated to the average T antigen levels (i.e., the expression potential).ConclusionsThese data show that Tag expression potential in these lines varies depending on the vector and clonal variation, but that the observed level depends on cell density and cell cycle transit time. The hypothetical terms, expression at zero cell density and expression at minimum G1 phase fraction, were introduced to simplify measures of expression potential.

Potential of adenoviral p53 gene therapy and irradiation for the treatment of malignant gliomas.

We investigated the combined effects of p53 gene transfer and irradiation and its still unclear interaction mechanism in human gliomas. Four human glioma cell lines expressing mutant type p53 (U373 and A172) and wild-type p53 (D54MG and EFC-2) were transfected by adenoviral vectors bearing p53 gene at 50 multiplicity of infection. Two days after transfection, cells were irradiated (3, 6, and 9 Gy). The cytotoxicity was evaluated by clonogenic assay. The quantitative analysis of apoptosis and cell cycle analysis were performed using flow cytometry. Irradiation combined with adenoviral p53 transfection significantly increased cytotoxicity, which was additive in cell lines with wild-type p53 and more than additive in cell lines with mutant p53. The combination of two modalities increased the apoptotic population by 14% in A172 cells and 20% in D54 MG cells, which were the sum of apoptosis from each modality. Adenoviral p53 transfection increased the G1 phase fraction and concomitant decrease of radioresistant S phase fraction in A172 and D54MG cells. Our study demonstrated that p53 gene transfer combined with irradiation increased absolute cytotoxicity in human glioma cells used in this experiment. The interaction mechanism for increased cytotoxicity involved, in part, increased apoptosis and change of cell cycle profile.

Influence of p53 and bcl-2 on proliferative activity and treatment outcome in head and neck cancer patients.

Knowledge about the influence of biomarkers on cell proliferative activity might explain differences in radiosensitivity between head and neck tumors and might improve patient selection for the most optimal treatment strategy. p53 and bcl-2 protein expression were determined immunohistochemically in 56 head and neck cancer patients, treated by surgery only in five cases and by radiotherapy, with or without surgery, in 51 cases. Relationships with various cell proliferation markers, determined by flow-cytometry (G1-phase fraction, S-phase fraction, BrdUrd-labeling index, duration of S-phase and potential doubling time) were investigated. Associations between these cell proliferation parameters, on the one hand, and both p53 and bcl-2, on the other, were not found. Furthermore, p53 and bcl-2 expression were both not related to clinicopathological parameters (T- and N-stage, site, grade) and did not affect loco-regional recurrence-free survival and/or disease-free survival. We could not find a prognostic value for both p53 and bcl-2 protein expression to differentiate radiosensitive from radioresistant head and neck tumors.

Expression of bcl-2 in Non-small Cell Lung Cancer and its Effects on Cell proliferation and Survival

BACKGROUND: Tumor growth is the net result of intrinsic proliferation and escape from active cell death. bcl-2 is a member of a new category of oncogenes that is not involved in influencing cell proliferation but is involved in regulating cell death(apoptosis). Based on this information, it seems to be reasonable to expect that there may be clinical prognostic significance of bcl-2 expression in non-small cell lung cancer. But its prognostic significance is not established. METHODS: To investigate the role of bcl-2 in lung cancer, we performed immunohistochemical stain of bcl-2 on 57 biopsy specimens from resected primary non-small cell lung cancer. Thereafter, flow cytometric cell cycle analysis was done. And we analyzed the correlation between bcl-2 expression, clinical parameters, S-, G1-phase fraction and survival. RESULTS: 1) bcl-2 were detected in 43.8% of total 57 patients(according to histology, squamous cancer 47%, adenocarcinoma 32%, according to TNM stage, I 28.6%, II 52.3%, III 45.5%. both differences were insignificant). 2) By using the flow cytometric analysis, mean S-phase fraction of bcl-2(+) and (-) group were 14.1(+/-7.8)%, 24.7(+/-10.5)%(p<0.005), mean G1 -phase fraction of bcl-2(+) and bcl-2(-) group were 75.5(+/-10.8)%, 65.5(+/-11.4)%(p<0.05). 3) 2yr, 3yr and 5yr survival and median survival time of bcl-2(+) group were 65%, 54%, 41%, 53months, and those of bcl-2(-) group were 71%, 52%, 46%, 37 months. (p>0.05, Kaplan-Meier, log rank) CONCLUSION: bcl-2 was detected in 43.8% of primary non-small cell lung cancer. The S-phase fraction of bcl-2(+) group was less than bcl-2(-) group, and G1 -phase fraction of bcl-2(+) group was more than bcl-2(-) group. But, expression of bcl-2 could not be a prognostic factor.

Increase in Expression of Hsp47 and Collagen in Hereditary Gingival Fibromatosis is Modulated by Stress and Terminal Procollagen N-Propeptides

HGF is a rare oral condition characterized by a slow, progressive enlargement of the gingiva, involving both the maxilla and mandible. HGF provides a model for the study of regulatory features of conditions characterized by connective tissue hyperplasia. In this study, the culture characteristics of gingival fibroblasts derived from patients of the same family with HGF (n = 4) were similar with regard to cell cycle analysis. Flow cytometric DN A content analysis revealed uniform DNA diploidy for fibroblasts cultured from NG and HGF. NG cells showed a low S-phase fraction (19.8%) and G2/M fraction (5.8%) and a relatively high G1 phase fraction (74%). In contrast, HGF cells from all members of the tested kindred, exhibited diploid cells with a higher S-phase (40.9%) and G2/M (10.1 %) fraction and a relatively low G1 phase fraction (40.9%). Furthermore, we demonstrated that the expression and production of Hsp47 parallels the increased levels of collagen secretion observed in HGF. In addition, we show that Hsp47 and collagen are coordinately regulated following stress via a feedback mechanism mediated by N-terminal procollagen propeptides. Utilizing confocal microscopy and antibodies directed against GST-fusion proteins encompassing the pro a 1(1) N-propeptide globular domain (NP1) (residues 23-108), it was apparent that this regulatory mechanism does not involve significant interaction with Hsp47's chaperoning of procollagen.

Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures.

It has been shown that some B-cell hybridomas secrete autocrine factors in vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle G1 phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G1 phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.