Flow Cytometric Cell Cycle Analysis Research Articles

AVitex agnus-castusExtract Inhibits Cell Growth and Induces Apoptosis in Prostate Epithelial Cell Lines

Extracts of Vitex agnus-castus fruits (VACF) are described to have beneficial effects on disorders related to hyperprolactinemia (cycle disorders, premenstrual syndrome). A VACF extract has recently been shown to exhibit antitumor activities in different human cancer cell lines. In the present study, we explored the antiproliferative effects of a VACF extract with a particular focus on apoptosis-inducing and potential cytotoxic effects. Three different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3) representing different disease stages and androgen responsiveness were chosen. The action of VACF on cell viability was assessed using the WST-8-tetrazolium assay. Cell proliferation in cells receiving VACF alone or in combination with a pan-caspase inhibitor (Z-VAD-fmk) was quantified using a Crystal Violet assay. Flow cytometric cell cycle analysis and measurement of DNA fragmentation using an ELISA method were used for studying the induction of apoptosis. Lactate dehydrogenase (LDH) activity was determined as a marker of cytotoxicity. The extract inhibited proliferation of all three cell lines in a concentration-dependent manner with IC (50) values below 10 microg/mL after treatment for 48 h. Cell cycle analysis and DNA fragmentation assays suggest that part of the cells were undergoing apoptosis. The VACF-induced decrease in cell number was partially inhibited by Z-VAD-fmk, indicating a caspase-dependent apoptotic cell death. However, the concentration-dependent LDH activity of VACF treated cells indicated cytotoxic effects as well. These data suggest that VACF contains components that inhibit proliferation and induce apoptosis in human prostate epithelial cell lines. The extract may be useful for the prevention and/or treatment not only of benign prostatic hyperplasia but also of human prostate cancer.

Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the cell cycle in granulosa and fibroblast cells of cattle

The cell cycle stage of donor cells is an important factor influencing developmental ability of nuclear transfer embryos. In the present experiment, cumulus and fibroblast cells of cattle were subjected to flow cytometric cell cycle analysis before being used in somatic cloning experiments. The following experimental groups were analyzed for each cell type: (1) actively dividing cells, (2) cells confluent for 4 days, (3) cells starved for 1, 2, 3 or 5 days. Using the propidium iodide flow cytometric assay, there were no significant differences ( P ≥ 0.05) in the percentage of cells in G0/G1 regardless of origin and type of cell, after confluency or serum starvation. Differences with the growing cells were found ( P ≤ 0.01). To determine what subset of cells in G0/G1 were in the G0 subphase of the cell cycle, an immunofluorescence analysis was conducted using monoclonal anti-PCNA antibodies in a FACS assay. There were not statistically significant differences in the percentage of cells that enter G0, between confluent and any starved group for either type of cells. Bovine fibroblast cells, confluent or serum starved for 3 days, were used in nuclear transfer experiments. A slight trend for a more desirable fusion rate in starved cells was detected, and embryo cleavage was greater in starved cells, however, in vitro development to blastocysts was similar between groups. Data indicate that prolonged culture of cells in the absence of serum does not imply a shift in the percentage of cells that enter G0/G1 or G0 alone, and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs, because there are negative effects such as apoptosis, associated with serum starvation.

Characterization of cell-cycle progression and growth of WB-F344 normal rat liver epithelial cells following gamma-ray exposure.

Apparently normal rat liver epithelial cells (WB-F344) have been widely used in studies pertaining to carcinogenesis. Ionizing radiation, a well known carcinogen, is known to perturb cell-cycle progression in a dose-dependent manner, thereby causing delay in cell proliferation. However, for WB-F344 cells, there is a paucity of such data, which are of substantial importance in understanding their radiation response. Here, the distribution of phases in the cell-cycle and the proliferation ability of WB-F344 cells are characterized at various time points after the cells have been irradiated with different doses of gamma-rays. After WB-F344 cells reached 100% confluence, they were trypsinized and suspended at 3.5 x 10(5) cells/ml in culture medium. Cells were irradiated in suspension with (137)Cs gamma-rays at doses from 1-10 Gy. After irradiation, 1 x 10(5) cells were plated into 60 x 15-mm culture dishes and incubated at 37 degrees C, with 2% CO(2) and 98% air. At 12, 24, 36, 48, and 60 h postirradiation, cells were harvested, counted, and subjected to flow cytometric cell-cycle analysis. Growth curves of WB-F344 cells irradiated with gamma-rays started to separate at 36 h postirradiation. By 60 h postirradiation, the growth curves for each of the 10 absorbed doses were distinctly separated. Drastic redistributions of control and irradiated cells within G(0)/G(1)-, S-, and G(2)/M-phases of the cell cycle were observed during the first 36 h of cell growth. At each time point postirradiation, cell-cycle phase profiles of irradiated cells were altered in a dose-dependent manner. In general, there was a strong correlation between the percentage of G(2)/M-phase cells and absorbed dose, with the exception of 24 h postirradiation. The percentage of G(2)/M-phase cells increased as a function of time postirradiation, suggestive of delays in the passage of cells through the G(2) cell-cycle checkpoint. This work provides a general description of cell cycle redistribution and repopulation kinetics of WB-F344 cells at various times postirradiation of quiescent cells that were subsequently allowed to proliferate. In general, growth inhibition and delays in progression through G(2)/M-phase correlated well with radiation dose. These data should be of considerable significance in the design of experiments that examine the radiation response of these cells.

Normal HC11 and ras-transformed mouse mammary cells are resistant to the antiproliferative effects of retinoic acid.

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 M 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of -casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR , as well as RXR a, and g expression was low in both HC11 and HC11ras cells. In addition, RAR mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

Flow cytometric cell-cycle analysis of cultured fibroblasts from the giant panda, Ailuropoda melanoleuca L.

In animal cloning, it is generally believed that the inactive diploid G(0)or G(1)stage of the cell cycle is beneficial to initiate cell-cycle coordination and reprogramming following transfer of the donor nucleus. Previous experiments have demonstrated that serum starvation results in quiescent cell stage. Some experiments show that the majority of cells in a fully confluent cell culture are also in an inactive G(1)stage. In order to provide more G(0)/G(1)stage cells for giant panda cloning, we carried out a flow cytometric analysis of the cell cycle of fibroblasts from the abdominal muscle of a giant panda at different passage numbers under different growth conditions, and after different periods of serum starvation. The percentage of G(0)+G(1)stage cells differed significantly under different growth conditions. Serum starvation effectively increased the percentage of G(0)+G(1)stage cells, and the cell cycle characteristics following serum starvation for varying periods of time differed with this and the initial confluency of the cultures. The data should help in choosing the optimal stage for preparing donor cells as well as increasing the potential cloning efficiency in our study of giant panda cloning.

Different role of saturated and unsaturated fatty acids in β-cell apoptosis

It is believed that free fatty acids contribute to the pathogenesis of type 2 diabetes in humans. We have recently shown that lipoapoptosis of human β-cells is specifically induced by saturated fatty acids while unsaturated had no effect. In the present study we tested the effect of co-incubation of different saturated and unsaturated free fatty acids on lipoapoptosis in β-cells. RIN1046-38 cells and isolated human β-cells were incubated with combinations of saturated fatty acids (palmitate, stearate) and mono- or polyunsaturated fatty acids (palmitoleate, oleate, and linoleate). Cells were incubated for 24–72 h with 1 mM fatty acids. All unsaturated fatty acids tested completely prevented palmitate- or stearate-induced apoptosis of rat and human β-cells as assessed by flow cytometric cell cycle analysis and TUNEL assay. This might suggest that apoptosis in vivo is predominantly determined by the content of unsaturated fatty acids in a mixed fatty acid pool.

Overexpression of E2F-1 leads to bax-independent cell death in human glioma cells

Gliomas are highly resistant to any kind of treatment. Multiple genetic abnormalities exist in gliomas indicating that effective gene therapy should be directed towards replacement of multiple rather than single genes. Bax is a protein of the Bcl-2 family that promotes apoptosis and functions as a tumor suppressor gene. The E2F family of transcription factors plays a pivotal role in the regulation of cell-cycle and cell-death related genes in gliomas. We examined the therapeutic potential of the simultaneous transfer of Bax and E2F molecules (1, 2 or 4) to gliomas. We used first generation E1A-deleted adenoviral vectors to transduce the E2Fs and Bax cDNAs. The recombinant adenoviral vector encoding bax uses the inducible Cre-loxP system to transduce the protein expression. Western blot analysis and immunofluorescence assays demonstrated high level of expression of the exogenous proteins. Trypan blue cell viability assays and flow cytometric cell-cycle analysis demonstrated an additive effect of these molecules to induce cell death via apoptosis. Western blot analysis showed that the ectopic expression of E2F-1 decreased the level of expression of Bax. These results indicate that E2F-1 and Bax have an additive anti-glioma effect when expressed simultaneously at high levels. Our data also suggest that Bax is not involved in the E2F-1-mediated apoptosis.

Array-Based Structure and Gene Expression Relationship Study of Antitumor Sulfonamides IncludingN-[2-[(4-Hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide andN-(3-Chloro-7-indolyl)-1,4-benzenedisulfonamide

Compounds from sulfonamide-focused libraries have been evaluated in cell-based antitumor screens using the COMPARE analysis with a panel of 39 human cancer cell lines and flow cytometric cell cycle analysis. Thus far, 2 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide (E7010)) and 3 (N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide (E7070)) have been selected from the collections as potent cell cycle inhibitors, which have progressed to clinical trials. Compound 2 is an orally active antimitotic agent disrupting tubulin polymerization, whereas compound 3 belongs to a novel class of antiproliferative agents causing a decrease in the S phase fraction along with G1 and/or G2 accumulation in various cancer cell lines. Because both compounds exhibited preliminary clinical activities in the phase I setting, we decided to examine further this series of oncolytic small molecules, particularly by using high-density oligonucleotide microarray analysis. The array data have enabled us to characterize these two classes of antitumor sulfonamides on the basis of gene expression changes, illuminating the essential pharmacophore structure and drug-sensitive cellular pathways for each class. Moreover, the dual character of 5 (N-(3-chloro-7-indolyl)-4-methoxybenzenesulfonamide (ER-67880)), resembling both 2 and 3, was revealed by array-based transcription profiling, though the 3-type profile of this molecule had not been apparent in the cell-based phenotypic screens. These results provide an example of the utility of structure and gene expression relationship studies in medicinal genomics.

Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0+G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8±2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1±6.2 and 77.8±5.7%, respectively), and in cycling cells (72.7±16.3 and 78.4±11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0+G1, and prolonged starvation significantly increased (P<0.05) percentages of cells in G2+M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0+G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0+G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P<0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

Apoptosis Plays a Distinct Role in the Loss of Precursor Lymphocytes during Zinc Deficiency in Mice

Lymphopenia is a characteristic of zinc deficiency, which is associated with massive loss of pre-B and pre-T cells from the primary lymphoid organs of zinc-deficient mice that have elevated serum corticosterone (CS). We examined whether this naturally elevated glucocorticoid level is associated with increased apoptotic loss of pre-T cells in the thymus of A/J and CAF1/J mice. In three experiments, partially atrophied thymuses were removed from 20 marginally zinc-deficient (ZD) young adult mice and cultured for 6 h in parallel with thymocytes prepared from 17 adequately fed mice. Thymocyte immunophenotyping combined with flow cytometric cell cycle analysis was used to identify the degree of apoptotic cell death among thymocytes of the two dietary groups, which were compared in the absence of in vivo phagocytosis. Apoptosis was enhanced 50-300% among pre-T cells (CD4+CD8+) prepared from ZD mice. This resulted in a 38% shrinkage of the thymic pre-T cell compartment, which was associated with an 80% decrease in thymic cell number. Pro-T cells (CD4-CD8-) and mature T cells (CD4+CD8-, CD4-CD8+), which express higher levels of Bcl-2 protein, survived ZD to a greater extent and formed a greater proportion of the remaining thymocyte population in ZD mice. Collectively, these data show that heightened degrees of apoptotic cell death induced in vivo by CS-disrupted thymic T cell lymphopoiesis, identifies the means of disruption of marrow B cell lymphopoiesis and explains the appearance of lymphopenia.

Flow Cytometry-A New Tool for Direct Control of Fermentation Processes

With flow cytometric cell cycle analysis, fermentation processes become transparent. This implies that we can control the process, by determining growth phases of the yeast population at specific times during the fermentation process, and that the process can be optimised and regulated directly.

Multivariate Analysis of the Prognostic Significance of DNA-Ploidy and S-Phase Fraction in Ovarian Cancer Determined by Flow Cytometry Following Detection of Cytokeratin-Labeled Tumor Cells

Objective. The outcome of patients with advanced ovarian cancer is poor despite aggressive therapy including surgery and multiagent chemotherapy. Valid prognostic factors are necessary to estimate the course of the disease and to define biologically similar subgroups for analysis of therapeutic efficacy.Methods. This study is the first published prospective study concerning the prognostic significance of DNA-ploidy and S-phase fraction in ovarian cancer following enrichment of tumor cells by cytokeratin labeling. Epithelial cells were labeled by FITC-conjugated cytokeratin antibody (CK 5, 6, 8, and 17) prior to flow cytometric cell cycle analysis in 129 fresh specimens of primary ovarian cancer.Results. Recurrence-free survival of patients with DNA-diploid primary ovarian cancer was significantly better compared to that of patients with DNA-aneuploid tumors in univariate analysis (47% vs 18%, P = 0.01). The tumor-dependent overall survival of patients with DNA-diploid tumors was 57% compared to 30% with DNA-aneuploid tumors (P = 0.04). In FIGO stage III ovarian cancer DNA-ploidy, optimized by cytokeratin gating for tumor cells, achieved independent prognostic significance. No significance was found for S-phase fraction. However, despite convincing methodological advantages in the detection of DNA-aneuploid subpopulations the clinical significance of cytokeratin gating of epithelial cells was only marginal.Conclusion. DNA-ploidy has been shown to be as powerful or even more so in comparison to postoperative residual tumor in multivariate analysis for predicting clinical outcome in advanced ovarian cancer. Thus, determination of DNA-ploidy should be introduced to currently recruiting phase III studies for therapy of ovarian cancer for better definition of prognostic subgroups.

Evaluation of prognostic factors following flow-cytometric DNA analysis after cytokeratin labelling: I. Breast cancer.

In gynecologic oncology valid prognostic factors are necessary to estimate the course of disease and to define biologically similar subgroups for analysis of therapeutic efficacy. The presented study is a prospective study concerning prognostic significance of DNA ploidy and S‐phase fraction in breast cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC‐conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17) prior to flow cytometric cell cycle analysis in 327 fresh specimens of primary breast cancer. Univariate analysis in breast cancer detected the prognostic significance of DNA‐ploidy, S‐phase fraction and CV (coefficient of variation) of G0G1‐peak of tumor cells for clinical outcome, especially for nodal‐negative patients. Multivariate analysis could not confirm prognostic evidence of DNA‐ploidy and S‐phase fraction. In conclusion, in breast cancer no clinical significance for determination of DNA‐parameters was found.

Evaluation of prognostic factors following flow-cytometric DNA analysis after cytokeratin labelling: II. Cervical and endometrial cancer.

In gynecologic oncology valid prognostic factors are necessary to define biologically similar subgroups for analysis of therapeutic efficacy. This study is the first published prospective study concerning prognostic significance of DNA ploidy and S‐phase fraction in cervical and endometrial cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC‐conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17) prior to flow cytometric cell cycle analysis in 91 specimens of cervical cancer and 73 samples of endometrial cancer. In cervical cancer neither DNA‐ploidy nor S‐phase fraction were relevant prognostic parameters. But CV of the G0G1‐peak showed prognostic relevance in cervical cancer cells, even in multivariate analysis. This interesting observation, however, seems to have no therapeutic consequence due to the small discrimination capacity of CV. In endometrial carcinoma, gross DNA‐aneuploidy (DNA‐index > 1.3) and a high percentage of proliferating cells (>75th percentile) were univariate and multivariate highly significant prognostic factors for recurrence‐free survival. Especially DNA‐aneuploidy (DI>1.3) is one of the most important independent molecular biological prognostic factors. While diagnostic curettage we could identify risk patients even preoperatively by determination of the prognostic factors like histologic tumor type, grading, cervical involvement and DNA‐ploidy. Thereby these patients could be treated primarily in an oncologic center. In conclusion, our investigations showed that the determination of DNA‐ploidy should be done in endometrial carcinoma. In cervical cancer no clinical significance for determination of DNA‐parameters was found.

Disruption of the p53 gene results in preserved trabecular bone mass and bone formation after mechanical unloading.

We tested the hypothesis that mechanical unloading facilitates signaling of p53, an important modulator of cell cycling and apoptosis, in bone marrow cells and thereby reduces trabecular bone volume (BV). We performed histomorphometric analyses and bone marrow cell cultures in tail-suspended (TS) p53 null (p53-/-) and wild-type (p53+/+) mice. Eight-week-old male mice were assigned to four groups after 1-week acclimatization: p53+/+ + ground control (GC), p53+/+ + TS, p53-/- + GC, and p53-/- + TS. Bilateral tibial samples were used for analysis. The histomorphometric parameters of trabecular structure, formation and resorption did not differ between the p53-/- + GC and p53+/+ + GC groups. Trabecular BV in p53+/+ + TS mice was significantly reduced to 45% of that in the p53+/+ + GC group after one week of TS. In contrast, BV in p53-/- + TS mice was preserved at the same level as that in the p53-/- + GC group. The bone formation rate (BFR) was significantly reduced in p53+/+ + TS but not in p53-/- + TS mice. Unloading significantly increased trabecular osteoclast number (Oc.N) and surface in p53+/+ + TS mice compared with the p53+/+ + GC group, but the difference was not significant between p53-/- + TS and p53-/- + GC mice. In bone marrow cell culture, the numbers of alkaline phosphatase-positive (ALP+) colony-forming units fibroblastic (CFU-f) and mineralized nodules were significantly reduced in p53+/+ + TS, but not p53-/- + TS mice. [3H]thymidine incorporation into bone marrow cells was higher in p53-/- mice than in p53+/+ mice, independent of mechanical loading or unloading. Flow cytometric cell cycle analysis revealed that unloading significantly increased the percentage of hypoploid bone marrow cells in p53+/+ mice relative to that in p53+/+ + GC mice, but there was no significant difference in ploidy between p53-/- + TS and p53-/- + GC mice. Expression levels of p53 and p21 mRNAs were enhanced after TS in bone marrow cells from p53+/+ mice. Our data show that trabecular bone mass and bone formation were preserved after tail-suspension in p53-/- mice, closely associated with ALP+ CFU-f and mineralized nodule formation in marrow cultures obtained from tibias of p53-/- mice. We speculate that bone loss due to mechanical unloading may be related to facilitation of intracellular p53-p21 signaling.

Antitumor activity- and gene expression-based profiling of ecteinascidin Et 743 and phthalascidin Pt 650

Background: Ecteinascidin 743 (Et 743) is a potent antitumor marine alkaloid currently undergoing phase II clinical trials. The synthetic analog phthalascidin (Pt 650), a designed structural analog of Et 743 displays in vitro potency comparable to Et 743. In this study, we used a panel of 36 human cancer cell lines, flow cytometry and oligonucleotide microarrays to analyze further these two compounds in a parallel fashion with regard to both antitumor activity (phenotype) and gene expression (genotype) bases. Results: The cancer panel experiment established that activity patterns of Et 743 and Pt 650 were essentially the same with their IC 50 values ranging from pM to low nM. By means of flow cytometric cell cycle analysis using HCT116 cells, they were shown to disrupt S phase progression after a 12-h treatment at 2.0 nM, eventually resulting in the late S and G2/M accumulation at the 24-h time point. Array-based gene expression monitoring also demonstrated that the Et 743 and Pt 650 profiles were highly similar in two distinct cancer cell lines, HCT116 colon and MDA-MB-435 breast. Characteristic changes were observed in subsets of genes involved in DNA damage response, transcription and signal transduction. In HCT116 carrying the wild-type p53 tumor suppressor gene, the up-regulation of several p53-responsive genes was evident. Furthermore, a subset of genes encoding DNA-binding proteins to specific promoter regions (e.g. the CCAAT box) was down-regulated in both cell lines, suggesting one potential mode of action of this series of antitumor agents. Conclusion: A combination of gene expression analysis using oligonucleotide microarrays and flow cytometry confirms an earlier finding that Et 743 and Pt 650 have remarkably similar biological activities.

Cell cycle analysis and detection of proliferative cell nuclear antigen of the endometrium after hormone replacement therapy

Background: To understand the effect of sequential combined hormone replacement therapy (HRT) on the postmenopausal endometrium. Methods: Sonographic endometrial thickness, endometrial histopathology, flow cytometric cell cycle analysis and the level of proliferative cell nuclear antigen (PCNA) were studied. Results: One hundred and thirty-eight postmenopausal women were enrolled in this study. Among which, 97 women had their endometrium being adequately obtained; the most frequent type of histopathology was normal endometrium (91.8%). Endometrial hyperplasia was found in seven patients (7.2%), including typical simple hyperplasia ( n=1, 1%), focal simple hyperplasia ( n=5, 5.2%) and complex hyperplasia without atypia ( n=1, 1%). The proliferative fractions (PF; S plus G 2–M phase) of cells from normal and hyperplastic endometrium of menopausal women after HRT were 8.18 and 8.95%, respectively, which were lower than those from 29 premenopausal women without HRT. The level of PCNA of normal and hyperplastic endometrium in postmenopausal women after HRT was about 80 and 84%, respectively, of that from premenopausal endometrium. Conclusions: Our study showed the PF of the cell cycle and the level of PCNA were not increased in the menopausal endometrium under HRT as compared to the premenopausal controls.

Co-expression of E2F-2 enhances the p53 anti-cancer effect in human glioma cells.

Gliomas are highly resistant to conventional treatment. Improved knowledge of the molecular defects of glioma cells offers new avenues for the development of gene therapy strategies. Transfer of the p53 gene has proven effective in suppressing proliferation in human glioma cell lines. However, several human glioma cell lines are resistant to p53-induced cell death. The E2F family of transcription factors are pivotal for the regulation of cell-cycle and cell-death related genes in gliomas. In the present study, we sought a more effective strategy for glioma treatment by examining the therapeutic potential of the simultaneous transfer of p53 and E2F-2 to gliomas. Trypan blue cell viability assays and flow cytometric cell-cycle analysis demonstrated that the transfer of both p53 and E2F-2 induced cell death in D-54 MG, a p53-resistant glioma cell line. In addition, transfer of E2F-2 did not interfere with the apoptotic properties of exogenous wild-type p53 in U-251 MG cells. Finally, the expression of E2F-2 in D-54 MG cells suppressed the expression of the apoptotic molecule mdm-2 induced by exogenous p53 in these cells. These results show that co-expression of E2F-2 and p53 enhances the anti-cancer effect of p53 in gliomas.

Flow cytometric DNA analysis using cytokeratin labeling for identification of tumor cells in carcinomas of the breast and the female genital tract.

Flow cytometric assessment of DNA‐ploidy and S‐phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC‐conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively) prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA‐aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA‐diploid tumors, a significantly improved detection of S‐phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating “normal cells”. S‐phase fraction following tumor cell enrichment was increased by 10% (mean) following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA‐analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.

Laser Capture Microdissection–Guided Fluorescence In Situ Hybridization and Flow Cytometric Cell Cycle Analysis of Purified Nuclei from Paraffin Sections

Laser capture microdissection (LCM) has recently been identified as a quick, simple, and effective method by which microdissection of complex tissue specimens for molecular analysis can be routinely performed. Assessment of gene copy number by fluorescence in situ hybridization (FISH) is useful for the analysis of molecular genetic alterations in cancer. Unfortunately, the application of FISH to paraffin sections of tumor specimens is fraught with technical difficulty and potential artifacts. Our results demonstrate that LCM-microdissected nuclei are suitable for FISH gene copy analysis. Amplification of genes in cancer specimens can be detected as easily in LCM-prepared nuclei as in fresh nuclei from cancer tissue specimens. Furthermore, contamination of tumor specimens by normal cells can make interpretation of flow cytometric cell cycle analysis difficult. Our results show that LCM-microdissected nuclei can also be used for flow cytometric cell cycle and ploidy analysis.LCM/FISH offers the advantages of multicolor FISH in a morphologically defined cell population, without the technical problems of FISH performed on paraffin sections. This technique should further simplify the methodology required to perform copy number analysis of tumor suppressor or proto-oncogenes in archived cancer specimens. The use of LCM specimens will also improve the specificity and simplify the interpretation of flow cytometric cell cycle and ploidy analysis of breast cancer specimens.