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Results for “Laser Scanning Cytometry”

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PubMed2003

Slide-based cytometry for predicting malignancy in solid salivary gland tumors by fine needle aspirate biopsies.

BACKGROUND: To minimize hospitalization and morbidity for a patient with a solid tumor of a salivary gland, malignancy must be confirmed or excluded as soon as possible. This information cannot be obtained preoperatively by existing standard procedures. Minimal-invasive approaches with adequate diagnostic analysis represent a promising precondition for optimized therapy. METHODS: For fine needle aspirate biopsies (FNABs), laser scanning cytometry (LSC) offers a semi-automated slide-based technology for objective and quantitative analysis. We have established an assay for FNABs from salivary gland tumors. FNAB cells were stained for cytokeratin and DNA followed by LSC analysis. The cells were subsequently HE-stained and were relocalized on the slide. The LSC analysis quantitatively determines the DNA index (DI) of the tumor cells taking leukocytes as internal DNA diploid standard. Histograms with 0.95 < DI < 1.05 and 1.9 2.5 (i.e., 5c exceeding rate, 5cER) was calculated. Samples with DNA aneuploid peaks or with 5cER > 5% were classified as malignant. Routine histopathology was performed as a control. RESULTS: FNABs from 51 solid salivary gland tumors (41 parotid gland, six submandibular, four parapharyngeal) were analyzed with this assay. Eleven of 14 malignant tumors were DNA aneuploid by LSC analysis. All benign tumors showed diploid DNA content. The positive predictive value for malignancy was 1.0, the negative predictive value was 0.93, the correlation with routine histopathology was highly significant (p = 7.6 x 10(-9), Fisher's exact test). The calculated specificity of LSC analysis was 1.0 and the sensitivity was 0.79. CONCLUSIONS: This pilot study demonstrates the validity of slide-based cytometry for the preoperative prediction of malignancy in solid tumors being inaccessible for incision biopsy but suitable for FNABs such as those of the parotid gland.

Adenoma, Pleomorphic↗

PubMed2005

Histone H2AX phosphorylation after cell irradiation with UV-B: relationship to cell cycle phase and induction of apoptosis.

Damage to DNA that engenders double-strand breaks (DSBs) triggers phosphorylation of histone H2AX on Ser-139. Expression of phosphorylated H2AX (gammaH2AX) can be revealed immunocytochemically; the intensity of gammaH2AX immunofluorescence (IF) measured by cytometry was reported to correlate with the frequency of DSBs induced by X-ray radiation or by DNA damaging antitumor drugs. The aim of the present study was to measure expression of gammaH2AX following exposure of HeLa and HL-60 cells to a wide range of doses of UV-B light (6.1 J/m(2)-3.45 kJ/m(2)) and using multiparameter flow and laser scanning cytometry (LSC) to correlate DNA damage with cell cycle phase and induction of apoptosis. In both cell lines, the highest degree of H2AX phosphorylation induced by UV was seen in S-phase cells, particularly during early portion of S. In cells that did not replicate DNA (G(1), G(2) and M) the degree of H2AX phosphorylation was markedly lower than that in S-phase cells, and was strongly UV dose-dependent. Furthermore, the level of UV-induced gammaH2AX in G(1), G(2) and M was much higher in HeLa- than in HL-60- cells. Apoptotic cells become apparent >2h after exposure to UV and exhibited nearly an order of magnitude higher intensity of gammaH2AX IF than that initially induced by UV; predominantly S-phase cells underwent apoptosis. While the suppression of DNA replication, by aphidicolin prevented the induction of H2AX phosphorylation by UV in most S phase cells, it had no effect on a small cohort of cells that appeared to be entering S-phase, that expressed very high levels of gammaH2AX. Furthermore, aphidicolin itself induced gammaH2AX in early-S phase cells. The induction of gammaH2AX by UV was inhibited, but the incidence of apoptosis increased, by 5 mM caffeine, a known inhibitor of PI-3-related kinases. The data are consistent with the notion that H2AX phosphorylation observed throughout S phase reflects formation of DSBs due to the collision of replication forks with the UV-induced primary DNA lesions. Induction of gammaH2AX in G(1), G(2) and M is likely a response to the primary DSBs generated during UV exposure and/or DNA repair. It is unclear why the latter process was more pronounced in HeLa than in HL-60 cells.

Aphidicolin↗

PubMed1998

Chromosome aberration assays in genetic toxicology testing in vitro.

The chromosome aberration test using cultured mammalian cells is one of the sensitive methods to predict environmental mutagens and/or carcinogens, and is a complementary test to the Salmonella/microsome assay (Ames test). From our recent survey of 951 chemicals which have been tested for their clastogenicity in cultured mammalian cells such as Chinese hamster fibroblasts or human lymphocytes, it was noted that 47% of them are consistently positive either with or without metabolic activation. When the test was performed using the cell line CHL/IU, 39.2% (292/745) were found to be positive. However, 8% (36/447) of such clastogens were positive only at an extremely high concentration of more than 10 mM. About 11% (48/447) of clastogens such as diethylstilbestrol (DES) and methyl AalphaC (Glob-P-1) induced mainly polyploid cells. Most chemicals induced chromatid-type aberrations, some induce only break-type aberrations at relatively high dose levels, but others induce more exchange-type aberrations at relatively low dose levels. Clastogenic activities were compared among different clastogens, using the D20 value, which is the minimum dose (mg/ml) at which aberrations were found in 20% of metaphases. In addition, the translocation (TR) value was calculated from the incidence of cells with exchange-type aberrations. It was suggested that possible carcinogens are included in the group of compounds with relatively low D20 values, but with high TR values. Karyological analysis was performed, using a FISH painting probe prepared from No. 1 chromosome of CHO cells, on the clonal subline isolated after treatment with benzo(a)pyrene. However, no specific changes common to the agent were detected. Laser scanning cytometry (LSC) was also applied to screen for abnormal karyotypes. A translocation between particular chromosomes was reflected by the deletion of a DNA peak.

Benzo(a)pyrene↗

PubMed1997

Multiple fucosyltransferases and their carbohydrate ligands are involved in spermatogenic cell-Sertoli cell adhesion in vitro in rats.

We have identified multiple fucosyltransferases (FTs) (alpha[1-2]-, alpha[1-3]-, alpha[1-4]-FTs) on cells of the rat seminiferous epithelium as demonstrated by fucose incorporation into phenyl-beta-D-galactoside (Ph-beta-D-Gal), 2'-fucosyllactose (2'-FL), and lacto-N-fucopentaose-l (LNF I), respectively. Now, using fluorescence laser scanning cytometry, we report that multiple FTs are implicated in germ cell-Sertoli cell adhesion in vitro. Sertoli cells were isolated from 19- to 21-day-old CD rats and cultured for 6-10 days. Mixed germ cells were obtained by enzymatic dispersion of adult rat testis and cultured overnight before labeling with 10 microM acetoxymethyl ester derivative of the fluorescent indicator, calcein. The adherent cell analysis and sorting 570 interactive laser cytometer was used to determine the number of labeled adherent germ cells on Sertoli cell monolayers in the presence or absence of a variety of low molecular weight acceptors for fucose. Coincubation of labeled germ cells with Sertoli cell monolayers in the presence of GDP-fucose, UDP-galactose, Ph-beta-D-Gal, 2'-FL, LNF I, and Lewis-X and 3'-sialyl-Lewis-X oligosaccharides resulted in significant reduction of germ cell binding when compared to that of the untreated controls or of control samples incubated with cellobiose, melibiose, and alpha-D-mannopyranose, which do not serve as fucose acceptors. Our results suggest that multiple FTs and their lectin/selectin ligands are involved in mediating germ cell-Sertoli cell adhesion to form a cohesive epithelium and thus aid germ cell adluminal translocation within the seminiferous epithelium.

Animals↗

PubMed2004

Mesenchymal stem cells can be differentiated into endothelial cells in vitro.

Human bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes, chondrocytes, and adipocytes in vivo and in vitro. The aim of this study was to investigate the in vitro differentiation of MSCs into cells of the endothelial lineage. MSCs were generated out of mononuclear bone marrow cells from healthy donors separated by density gradient centrifugation. Cells were characterized by flow cytometry using a panel of monoclonal antibodies and were tested for their potential to differentiate along different mesenchymal lineages. Isolated MSCs were positive for the markers CD105, CD73, CD166, CD90, and CD44 and negative for typical hematopoietic and endothelial markers. They were able to differentiate into adipocytes and osteocytes after cultivation in respective media. Differentiation into endothelial-like cells was induced by cultivation of confluent cells in the presence of 2% fetal calf serum and 50 ng/ml vascular endothelial growth factor. Laser scanning cytometry analysis of the confluent cells in situ showed a strong increase of expression of endothelial-specific markers like KDR and FLT-1, and immunofluorescence analysis showed typical expression of the von Willebrand factor. The functional behavior of the differentiated cells was tested with an in vitro angiogenesis test kit where cells formed characteristic capillary-like structures. We could show the differentiation of expanded adult human MSCs into cells with phenotypic and functional features of endothelial cells. These predifferentiated cells provide new options for engineering of artificial tissues based on autologous MSCs and vascularized engineered tissues.

Adipocytes↗

PubMed2006

Induction of centrosome amplification in p53 siRNA-treated human fibroblast cells by radiation exposure.

Centrosome amplification can be detected in the tissues of p53(-/-) mice. In contrast, loss of p53 does not induce centrosome amplification in cultured human cells. However, examination of human cancer tissues and cultured cells has revealed a significant correlation between loss or mutational inactivation of p53 and occurrence of centrosome amplification, supporting the notion that p53 mutation alone is insufficient to induce centrosome amplification in human cells, and that additional regulatory mechanisms are involved. It has recently been shown that gamma irradiation of tumor cells induces centrosome amplification. However, the precise mechanism of radiation-induced centrosome amplification is not fully understood. In the present study, CCD32SK diploid normal human fibroblasts were transfected transiently with short interfering RNA (siRNA) specific for human p53 (CCD/p53i). There was a small increase in the frequency of centrosome amplification in CCD/p53i cells (4.0%) without irradiation. In contrast, CCD/p53i cells after 5-Gy irradiation showed a marked increase in abnormal nuclear shapes and pronounced amplification of centrosomes (46.0%). At 12 h after irradiation, irradiated CCD/p53i cells were arrested in G(2) phase. By laser scanning cytometry, abnormal mitosis with amplified centrosomes was observed frequently in the accumulating G(2)/M population at 48 h after irradiation. In the present study, we found that siRNA-mediated silencing of p53 in normal human fibroblasts, together with DNA damage by irradiation, efficiently induced centrosome amplification and nuclear fragmentation, but these phenomena were not observed with either siRNA-mediated silencing of p53 or irradiation alone.

Cell Division↗

PubMed1998

Critical aspects in the analysis of apoptosis and necrosis.

Despite that large number of methods to analyze cell death, in particular apoptosis, that have been developed, identification of the mode of cell death and its quantitation is not always simple or straightforward. The difficulties, potential pitfalls and traps in quantitation of dead cells, whether apoptotic or necrotic are reviewed. The following are common flaws in the measurement of cell death, which include incorrect assumptions, erroneous data interpretation and shortcomings of the methodology: 1) Misclassification of apoptotic bodies or chromatin fragments as individual apoptotic cells based on cellular DNA content analysis by flow cytometry; 2) Assumption that the quantity of fragmented DNA extracted from cells represents the frequency of apoptosis; 3) Conjecture that the apoptotic index represents the cell death rate; 4) Assumption that apoptotic cells must exhibit classical features of apoptosis e.g. internucleosomal DNA fragmentation; 5) Inadequacy of methods that presume to discriminate between late apoptotic and necrotic cells; 6) Possibility of a selective enrichment or loss of apoptotic cells during cell separation on density gradients, during trypsinization or other procedures of cell collection; and 7) Inability to distinguish between live, nonapoptotic cells phagocytizing apoptotic bodies and genuine apoptotic cells by flow cytometric methods. Many of the problems stem from the difficulty in identifying apoptotic or necrotic cells. Because apoptosis and necrosis have been originally defined based on morphological criteria it is essential to confirm the mode of cell death by microscopy. Laser scanning cytometry (LSC), which combines the advantages of flow and image cytometry, offers the possibility of morphological examination of apoptotic cells. By virtue of this attribute LSC appears to be the instrument of choice for analysis of apoptosis.

Apoptosis↗

PubMed2004

Histone H2AX phosphorylation induced by selective photolysis of BrdU-labeled DNA with UV light: relation to cell cycle phase.

BACKGROUND: The induction of DNA double-strand breaks (DSBs) in chromatin triggers histone H2AX phosphorylation (on Ser-139) by ATM-, ATR-, or DNA-dependent protein kinases (DNA-PK). Phosphorylated H2AX, denoted as gammaH2AX, can be detected immunocytochemically using an antibody that is specific to the Ser-139-phosphorylated epitope. We previously reported that the induction DSBs by DNA topoisomerase I or II inhibitors can be monitored in individual cells by measuring gammaH2AX immunofluorescence (IF) by cytometry. The present study explored whether the detection of gammaH2AX IF can serve as a marker of the presence of the DNA precursor bromodeoxyuridine (BrdU) that is incorporated into DNA. METHODS: HeLa cells growing on microscope slides were incubated with BrdU for 1 h, rinsed free of the precursor, and incubated for different periods for up to 12 h. The cells were then briefly incubated with Hoechst 33342 (to sensitize BrdU-labeled DNA to ultraviolet [UV] light), irradiated with 300 nm UV light to photolyze BrdU-labeled DNA, transferred back into culture for an additional hour, and fixed. Cells were concurrently immunostained for gammaH2AX (Alexa Fluor 633) and cyclin A (fluorescein isothiocyanate); their DNA was counterstained with 4,6-diamidino-2-phenylindole. The intensities of cellular far red (gammaH2AX), green (cyclin A), and blue (DNA) fluorescences were measured by laser scanning cytometry. RESULTS: After a 1-h pulse of BrdU followed by exposure to UV, nearly all cells with S-phase DNA content had many-fold higher gammaH2AX IF than G(1) or G(2)/M cells. The nonirradiated cells had minimal ("programmed") expression of gammaH2AX, whereas the irradiated cells incubated without BrdU had uniformly elevated levels of gammaH2AX IF independent of the cell cycle phase. Pulse-chase experiments showed that the cohort of BrdU-labeled (gammaH2AX-positive) cells progressed through G(2)/M and into G(1) phase after 8 and 12 h of growth in BrdU-free medium, respectively. Bivariate analysis of gammaH2AX versus cyclin A expression for the gated S-phase cells showed a correlation between these variables, suggesting that the rate of BrdU incorporation (DNA replication) correlates with expression of cyclin A. CONCLUSIONS: Photolysis of BrdU-labeled DNA induces DSBs and leads to H2AX phosphorylation. Detection of gammaH2AX IF indicates the presence of the incorporated BrdU, is compatible with concurrent detection of other intracellular antigens, and can be used to demonstrate cell cycle kinetics.

Benzimidazoles↗

PubMed1999

Cytogenetic alterations in pituitary adenomas detected by comparative genomic hybridization.

Pituitary adenomas are benign monoclonal tumors that are either hormonally functional or nonfunctional. Although their histologic and immunocytologic characteristics have been studied extensively, cytogenetic studies are scarce. We have investigated the cytogenetic alterations and DNA ploidy patterns of 12 sporadic pituitary adenomas, including 2 growth-hormone-secreting tumors, 1 prolactinoma, and 9 nonfunctional adenomas, by comparative genomic hybridization (CGH) and laser scanning cytometry (LSC). CGH revealed that the mean number of sites of copy gain was significantly higher in functioning adenomas than in nonfunctioning tumors (P < 0.01). The most frequent change detected was loss of 13q (5 cases), with a minimal common overlapping region at 13q14. These findings suggest that a putative tumor suppressor gene on 13q14 may play an important role in the development of pituitary adenomas. DNA aneuploidy was detected by LSC in 3 of the 12 cases. The DNA aneuploid adenomas showed cytogenetic changes more frequently than did the DNA diploid tumors (P < 0.02).

Adenoma↗

PubMed2006

A quantitative study of the effects of ionizing radiation on endothelial cells and capillary-like network formation.

The initial events of angiogenesis comprise endothelial cell activation, migration, and proliferation. The characteristics of retinal endothelial cells and capillaries are significantly altered in a number of diseases including cancer. Since radiation has been shown as a useful tool in radiotherapy by altering the proliferative changes, it is important to evaluate the responses of the endothelial cells and the capillary network to radiation. We quantified functional and kinetic responses of endothelial cells and capillaries to radiation in an in vitro model. An in vitro angiogenesis model was introduced in our study with endothelial cells cultured on an extracellular matrix gel in which hollow tube-like structures could be rapidly formed. Vessel formation was quantified using stereological techniques. The cell cycle kinetics of endothelial cells and accumulation of DNA damage after radiation were measured using laser scanning cytometry. To study the response of proliferative capillary-like structures to radiation, the vessel network was irradiated with 2 gray (Gy). To evaluate functional and kinetic responses and differentiation of endothelial cells to radiation, cells were irradiated with 2 and 6 Gy. Progressive time- and dose-dependent loss of endothelial cells occurred starting 24 hours after radiation. Vessel growth was significantly retarded at the higher dose. A significant percentage of DNA breaks were detected dose-dependently. A large percentage of G1 cells were measured in the irradiated endothelial cell population when compared to the respective sham-treated control population. These results indicate that radiation-induced endothelial cell injuries destroy the integrity of vascular structure. We postulated that apoptosis may represent a biologically relevant mechanism of radiation-induced endothelial cell damage.

Animals↗

PubMed2003

Hormone-refractory breast cancer remains sensitive to the antitumor activity of heat shock protein 90 inhibitors.

PURPOSE: The antiestrogen tamoxifen (Tam) has been used as therapy against estrogen receptor (ER)-positive breast cancer for decades. Most tumors respond initially, but resistance frequently develops. The ER exists in a multiprotein complex containing the molecular chaperone heat shock protein (Hsp) 90, which is known to regulate the stability and activity of this receptor. Therefore, we investigated a ligand-independent approach to hormonal therapy that depletes cellular levels of the receptor by inhibiting the function of Hsp90. EXPERIMENTAL DESIGN: The activity of the Hsp90 inhibitor geldanamycin (GA) and its clinically relevant derivative, 17-allylamino-17-demethoxygeldanamycin (17AAG), was examined at the molecular and cellular levels using Tam-resistant MCF-7 breast cancer cells both in vitro and in tumor xenografts. RESULTS: The ER was depleted by GA in several Tam-resistant cell lines, as were other Hsp90 client proteins such as Akt and Raf-1. Unexpectedly, Tam inhibited ER depletion by GA but had no effect on destabilization of Akt or Raf-1. When SCID mice supplemented with Tam were treated with 17AAG, their tumors also showed no decrease in ER levels as measured by immunofluorescent staining and laser scanning cytometry. In these same tumors, however, decreased Akt and Raf-1 levels were observed. Drug administration also led to inhibition of tumor xenograft growth. The mechanism by which Tam inhibits GA-mediated ER depletion is unclear, but immunoprecipitation experiments showed that Tam does not inhibit the ability of GA to alter the ER-chaperone complex. CONCLUSIONS: Based on its ability to deplete the ER as well as other critical signaling molecules in Tam-resistant breast cancer, 17AAG may provide a useful alternative treatment for patients with recurrent, hormone-refractory breast cancer that should be explored further in Phase II trials. In this context, combined treatment with 17AAG and Tam should be avoided because Tam may inhibit the ability of 17AAG to deplete the ER, potentially reducing its anticancer activity.

Animals↗

PubMed2004

Gadd45 proteins induce G2/M arrest and modulate apoptosis in kidney cells exposed to hyperosmotic stress.

Gadd45 proteins are induced by hyperosmolality in renal inner medullary (IM) cells, but their role for cell adaptation to osmotic stress is not known. We show that a cell line derived from murine renal IM cells responds to moderate hyperosmotic stress (540 mosmol/kg) by activation of G(2)/M arrest without significant apoptosis. If the severity of hyperosmotic stress exceeds the tolerance limit of this cell line (620 mosmol/kg) apoptosis is strongly induced. Using transient overexpression of ectopic Gadd45 proteins and simultaneous analysis of transfected versus non-transfected cells by laser-scanning cytometry, we were able to measure the effects of Gadd45 super-induction during hyperosmolality on G(2)/M arrest and apoptosis. Our results demonstrate that induction of all three Gadd45 isoforms inhibits mitosis and promotes G(2)/M arrest during moderate hyperosmotic stress but not in isosmotic controls. Furthermore, all three Gadd45 proteins are also involved in control of apoptosis during severe hyperosmotic stress. Under these conditions Gadd45gamma induction strongly potentiates apoptosis. In contrast, Gadd45alpha/beta induction transiently increases caspase 3/7 and annexin V binding before 12 h but inhibits later stages of apoptosis during severe hyperosmolality. These results show that Gadd45 isoforms function in common but also in distinct pathways during hyperosmolality and that their increased abundance contributes to the low mitotic index and protection of genomic integrity in cells of the mammalian renal inner medulla.

Animals↗

PubMed2005

Route and type of nutrition influence nuclear factor kappaB activation in peritoneal resident cells.

Morbidity of intra-abdominal abscess is increased when severely injured patients are fed parenterally. Lack of enteral nutrition appears to impair peritoneal cavity host defense. Because the transcription factor nuclear factor kappaB (NFkappaB) regulates various genes involved in inflammatory responses and its activation is important for host defense, we hypothesized that enteral nutrition would preserve appropriate NFkappaB activation in peritoneal resident cells (PRCs), the first defense line against peritoneal contamination. Mice (n = 105) were randomized to chow (n = 38), intravenous (IV)-total parenteral nutrition (TPN) (n = 34), or intragastric (IG)-TPN (n = 33) for 5 days' feeding. In experiment 1, PRCs were harvested for measurement of intranuclear NFkappaB activity with or without in vitro lipopolysaccharide (LPS) stimulation using laser scanning cytometry and enzyme-linked immunoabsorbant assay. PRC numbers tended to be higher in enterally fed mice than in IV-TPN mice. The main PRC subpopulation was macrophages in all groups. NFkappaB activation was increased in response to LPS in chow mice, whereas there was no increase in the IV-TPN group. IG-TPN mice demonstrated moderate NFkappaB activation. In experiment 2, mice underwent cecal ligation and puncture (CLP). Survival was observed up to 5 days. In another set of mice, tumor necrosis factor (TNF) alpha levels of peritoneal lavaged fluid were measured 4 h after CLP. Survival times after CLP improved in the chow and IG-TPN groups compared with the IV-TPN group. TNFalpha levels were significantly higher in the chow than in the IV-TPN group. In conclusion, parenteral nutrition decreases PRC number and blunts NFkappaB activation in PRCs. These changes may impair host defense in the peritoneal cavity.

Active Transport, Cell Nucleus↗

PubMed2001

Preferential loss of 5q14-21 in intestinal-type gastric cancer with DNA aneuploidy.

BACKGROUND: Little is known about the genetic changes associated with DNA ploidy in gastric cancer (GC). The aim of this study was to identify recurrent or specific chromosomal regions of DNA sequence copy number aberrations (DSCNAs) that might harbor genes associated with DNA aneuploidy in GC. METHODS: We analyzed DSCNAs with comparative genomic hybridization and DNA ploidy by laser scanning cytometry in 16 primary intestinal-type GCs. RESULTS: All GCs examined showed at least one DSCNA (loss or gain); eight were DNA diploid (DD) tumors and eight were DNA aneuploid (DA) tumors. The frequent (>30%) DSCNAs were loss of 5q14-21 and gains of 7p11-14, 8q, 20q, and Xq25-26. Recurrent amplifications (>10%) were detected at chromosomal regions 6p, 7p, and 13q. The overall number of DSCNAs was significantly greater in DA than in DD tumors (P = 0.006). Furthermore, the number of aberrations was clearly greater with 5q loss than without 5q loss (P = 0.002). Losses of 5q14-21, 9p21-pter, 16q, and 18q21-qter were preferentially detected in DA tumors. CONCLUSION: The present observations indicate that there is a close relationship between DSCNA and DNA ploidy in intestinal-type GC and that gene(s) at 5q14-21, 9p21-pter, 16q, and/or 18q21-qter may play important roles in acquisition of DNA aneuploidy.

Adult↗

PubMed2000

Intranuclear localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma.

OBJECTIVE: To investigate, with laser scanning cytometry (LSC), proliferating cell nuclear antigen (PCNA) expression during the cell cycle in renal cell carcinoma. STUDY DESIGN: DNA ploidy and intracellular localization of PCNA in renal cell carcinoma were determined using LSC and immunohistochemistry. The subjects were nine patients who had received surgery for renal cell carcinoma. After DNA ploidy analysis, the glass slides were restained by immunohistochemistry of PCNA. LSC allowed direct observation of PCNA localization during the cell cycle because we could obtain immunohistochemical staining of PCNA as a function of cell cycle phase for individual cells. RESULTS: PCNA was not demonstrated in the nuclei of G0/G1 cells. PCNA expression increased from the S phase of the cell cycle. PCNA rapidly degraded at the end of the G2 phase. In the late G2 and M phase, PCNA was not detected in almost any nucleus. CONCLUSION: LSC allows morphologic observation of the intracellular distribution of PCNA during the cell cycle in renal cell carcinoma.

Adult↗

PubMed2004

Cell cycle-dependent expression of thyroid hormone receptor-beta is a mechanism for variable hormone sensitivity.

Thyroid hormone receptors (TRs) are ligand-regulatable transcription factors. Currently, little is known about the expression of TRs or other nuclear hormone receptors during the cell cycle. We thus developed a stable expression system to express green fluorescent protein-TRbeta in HeLa cells under tetracycline regulation, and studied TR expression during the cell cycle by laser scanning cytometry. Only approximately 9-15% of the nonsynchronized cell population expressed TR because the majority of cells were in G(1) phase and did not express detectable amounts of TR. However, when cells were synchronized in early S phase with hydroxyurea and then released, TR expression levels increased in a cell cycle-dependent manner and peaked to 30-40% cells expressing TR at late G(2)/M phase before declining to nonsynchronized levels. Moreover, we observed a direct correlation between transcriptional activity and TR expression during the cell cycle. Similar cell cycle-dependent findings also were observed for endogenous TR in rat pituitary GH(3) cells. Last, cycloheximide studies demonstrated that the increase in TR expression was primarily due to increased translation. These novel observations of cell cycle-dependent expression of TR suggest that differential hormone sensitivity can occur during the cell cycle and may contribute to cell cycle progression during normal development and oncogenesis.

Animals↗

PubMed1998

Ozone alters the distribution of beta1 integrins in cultured primate bronchial epithelial cells.

The effects of 0.5 ppm ozone exposure for 6 h on the synthesis and distribution of beta1 integrins were examined in bronchial epithelial cells cultured at an air-cell interface. Ozone exposure damaged cilia and caused significant cell loss. Immunocytochemical localization and quantification of the beta1 subunit in the remaining attached cells using scanning laser cytometry demonstrated time-dependent changes in beta1 distribution in response to ozone. Although no changes were detected immediately after exposure, beta1 immunoreactivity increased 23 +/- 5% and 66 +/- 6% at 6 and 24 h, respectively. The increased immunostaining was localized at the apical surfaces and, to a lesser extent, at cell-cell contacts of cultured cells. Furthermore, integrin redistribution was not due to increased messenger RNA (mRNA) levels and protein synthesis because levels of beta1 mRNA and newly synthesized beta1 protein did not change after ozone exposure. However, immunoprecipitation analysis of beta1 integrins in lysates from equal numbers of cells showed that ozone-exposed cells contained 90 +/- 15% more total beta1 subunit at 24 h after exposure. In addition, our results demonstrated the presence of the alpha5beta1 integrin complex in bronchial epithelial cells and that the detergent-soluble amount of its associated beta1 subunit increased 60 +/- 10% in lysates of ozone-exposed cells. In conclusion, ozone altered cellular distribution of beta1 integrins in the remaining attached cells subsequent to cell injury and loss. The changes in beta1 distribution might be due to increased detergent extractibility of beta1 integrins rather than a real increase in the synthesis of beta1 integrins.

Animals↗

PubMed2000

Activity of cisplatin and ICI 182,780 on estrogen receptor negative ovarian cancer cells: cell cycle and cell replication rate perturbation, chromatin texture alteration and apoptosis induction.

The activity of cisplatin (CP, range of concentrations 0.25-1 microg/ml), the pure steroidal antiestrogen compound ICI 182,780 (range of concentrations, 0.01-10 microM) and various combinations of, was investigated on an estrogen receptor negative ovarian cancer cell line (A2780 WT) and its CP-resistant derivative subline (A2780 CP3). CP markedly reduced A2780 WT cell growth but marginally affected A2780 CP3, whereas ICI 182,780 was effective on both cell lines. CP but not ICI 182,780 provoked a significant blockade in late S/G(2) phase in both cell lines, particularly in the parental line. Measuring the number of rounds of cell replications showed that CP diminished the cell replication rate of both cell lines, particularly in A2780 WT. Conversely, ICI 182,780 reduced the cell replication rate of A2780 CP3 but not A2780 WT cells. Both drugs provoked apoptosis in A2780 WT cells, as assessed by the appearance of large (50-300 kbp) DNA fragmentation. However, laser scanning cytometry showed that only CP induced a measurable alteration of chromatin texture in A2780 WT but not in A2780 CP3 cells. The combination CP and ICI 182,780 resulted in a synergistic inhibitory activity of cell growth with a CP potentiation up to 4 and 11-fold in A2780 WT and A2780 CP3 cells, respectively. This reflected an enhanced reduction of the cell replication rate and did not involve perturbations of the cell cycle other than those provoked by CP alone. Apoptosis induction and the level of CP-DNA adducts were not influenced by adding ICI 182,780 to CP in both cell lines.

Antineoplastic Combined Chemotherapy Protocols↗