Cell Cycle Distribution Pattern Research Articles

Geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro.

Geldanamycin (GA) is an antibiotic targeting the ADP/ATP binding site of heat shock protein 90 (Hsp90). In screening for anti-herpes simplex virus type 1 (HSV-1) candidates, we found GA active against HSV-1. HSV-1 replication in vitro was significantly inhibited by GA with an 50% inhibitory concentration of 0.093 microM and a concentration that inhibited cellular growth 50% in comparison with the results seen with untreated controls of 350 microM. The therapeutic index of GA was over 3700 (comparable to the results seen with acyclovir). GA did not inhibit HSV-1 thymidine kinase. Cells infected with HSV-1 demonstrated cell cycle arrest at the G(1)/S transition; however, treatment with GA resulted in a cell cycle distribution pattern identical to that of untreated cells, indicating a restoration of cell growth in HSV-1-infected cells by GA treatment. Accordingly, HSV-1 DNA synthesis was suppressed in HSV-1(+) cells treated with GA. The antiviral mechanism of GA appears to be associated with Hsp90 inactivation and cell cycle restoration, which indicates that GA exhibits broad-spectrum antiviral activity. Indeed, GA exhibited activities in vitro against other viruses, including severe acute respiratory syndrome coronavirus. Since GA inhibits HSV-1 through a cellular mechanism unique among HSV-1 agents, we consider it a new candidate agent for HSV-1.

Effects of high cell density on growth-associated monoclonal antibody production by hybridoma T0405 cells immobilized in macroporous cellulose carriers

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production of hybridoma T0405 cells immobilized in macroporous cellulose carriers were investigated in continuous and batch cultures. The results showing, that the specific MAb production rate increased with increasing specific growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. Moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomena, MAb mRNA expression and cell cycle distribution were investigated in batch cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromode-oxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb mRNA expression reached the peak during the exponential growth phase, suggest a positively growth-associated MAb production. And the immobilized cells continued the MAb mRNA expression until dead phase, which was longer than that in suspended cells. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated MAb productivity of T0405 cells.

Effects of UFT (mixed compound of tegafur and uracil) on cell kinetics and inhibition of thymidylate synthase in L1210 ascites tumor.

Previous work in our laboratory showed that UFT (mixed compound of tegafur and uracil, molar ratio 1:4, respectively) caused the prolonged reduction of dTTP in L1210 leukemia cells in comparison with 5-fluorouracil (5-FU). The purpose of this study was to assess the effect of UFT on cell cycle distribution and thymidylate synthase activity of a leukemia cell line as compared with 5-FU. UFT and 5-FU were orally given to BDF1 mice bearing L1210 ascites tumor on day 3 after the tumor inoculation. Cell cycle distribution patterns at 24 hr after the drug administration showed a higher percentage of S phase in tumor cells treated with UFT than in those treated with 5-FU. Until 6 hr after the oral administration of the drugs, UFT inhibited the incorporation of [3H] deoxyuridine into DNA more long than 5-FU did. These results indicated that UFT has longer and stronger inhibitory effects on DNA replication than 5-FU in vivo under the employed experimental conditions (i.e., low and single doses of these fluorinated pyrimidines).

Image analysis of in situ cell cycle related changes of PCNA and Ki‐67 proliferating antigen expression

Abstract. This study reports on the proliferating cell nuclear antigen (PCNA) and Ki‐67 cell cycle related expression and distribution pattern analysed in the same cells. MCF‐7 cells were synchronized by mitotic detachment and triple stained for DNA, PCNA and Ki‐67. The major cell type was identified on each time sample as a function of the PCNA/Ki‐67 pattern, and both antigens as well as DNA were quantified. During G1 phase, the expression of PCNA greatly increased whereas Ki‐67 content decreased. During S phase, nuclear Ki‐67 content continuously increased especially in the second half of this phase, mainly due to the accumulation of the antigen in the nucleoli. During G2 phase, the antigen significantly passed into the nucleoplasm, its content continued to increase and reached its maximum in mitotic cells. Nuclear PCNA content mostly increased in the first part of S phase and sharply declined in mitotic cells as the antigen shifted to the cytoplasm. Cells showing PCNA positive Ki‐67 negative labelling were observed in all time samples from the beginning of the experiment. Their nuclear size, DNA content (of G1 cells), PCNA content (equivalent to the content of some late G, cells) and time occurrence (their percentage increased after the last late G1 cells had disappeared) tend to indicate that these cells have left the cycle by the end of G1 phase to enter a quiescent state. Cells coming out of mitosis split into two groups according to their Ki‐67/PCNA content. The biggest fraction was PCNA negative and Ki‐67 positive while the smallest showed positive staining for both antibodies. Cells of this second cohort slowly lost their 1–67 while their PCNA content increased as they moved through G1. Concurrently, most of the cells of the first cohort (here called Q2 and Q3 cell types) lost their Ki‐67 without increasing their PCNA content; then they joined cells of the second cohort by increasing their PCNA content at the end of G, phase. Some cells of this first cohort can also increase their PCNA and thus reach cells of the first cohort before the end of G1 phase. The existence of these two main cell cohorts suggests that cells after mitosis differ in some way that make them progress dlfferently through G1. Some cells seem to go through early G1 (G1a and late G1 (Glb) while others may come out of mitosis committed to go through the following cycle by directly entering late G1 compartment.

Characterisation of a tamoxifen-resistant variant of the ZR-75-1 human breast cancer cell line (ZR-75-9a1) and ability of the resistant phenotype.

A 6-month exposure of ZR-75-1 human breast cancer cells to tamoxifen (1 microM rising to 2 microM). resulted in a fall in oestrogen receptor (ER) levels from 225 fmol mg protein-1 to 56 fmol mg protein-1 while progesterone receptor (PGR) concentration fell from 63 fmol mg protein-1 to undetectable levels. Sensitivity to the anti-proliferative effects of tamoxifen was unchanged. A further 6 months' exposure to 4 microM tamoxifen resulted in loss of detectable ER and PGR and development of resistance to tamoxifen. Resistant cells, designated ZR-75-9a1, displayed morphological changes consistent with the acquisition of a less well differentiated phenotype. Flow cytometric studies demonstrated that the cell cycle distribution pattern of the resistant variant growing in the presence of 8 microM tamoxifen was identical to that of the untreated parent line, which showed marked accumulation of cells in G0/G1 when exposed to 8 microM tamoxifen. The resistant phenotype was not stable if cells were transferred to complete drug-free medium, but remained stable for at least 3 months in the presence of medium lacking oestrogenic activity. ZR-75-9a1 cells differ from previously reported tamoxifen-resistant variants of the MCF-7 line which retain ER and may prove a valuable model for the study of the development and stability of tamoxifen resistance in human breast cancer.

Comparative morphological and growth kinetics studies of human hair bulb papilla cells and root sheath fibroblasts in vitro

Dermal papillae isolated from anagen hair bulbs obtained from biopsy specimens from five subjects with normal hair pattern, and fibroblasts derived from the mesenchymal root sheaths (RSF) of the same hair follicles were separately grown in culture and the cell-cycle distribution pattern on different days was analyzed by applying DNA flow cytometry (FCM). Papilla cells (PC) exhibited distinctive morphological features by forming cell aggregates differing from RSF with respect to cell shape and growth pattern. They also proliferated remarkably more slowly than RSF. DNA-FCM analysis showed that both PC and RSF demonstrated synchronous fluctuations in the percentage of cells in G1/0, S and G2 + M phases during the period of subculture.

Cell cycle distribution patterns and generation times of L929 fibroblast cells persistently infected with Coxiella burnetii.

Coxiella burnetii established a persistent infection of various cell lines including L929 mouse fibroblasts. Although the basis for such persistence is unknown, the phenomenon does require continual growth, proliferation, and maintenance of viability of the host cells. We examined the effect of short- and long-term infection on the host cell's generation time and cell cycle. Flow cytometric studies of actively growing normal and infected cells stained with mithramycin or propidium iodide revealed no significant difference in cell cycle distribution patterns or changes in ploidy level associated with persistent infection with either phase I or phase II C. burnetii. The population doubling times of infected and normal cells were similar.