Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de-adaptation of EUE cells to a hypertonic medium

EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle-related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/1 range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the G0/1 range of DNA contents: this suggests that a G0 block occurs under long-term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.     

Dual parameter flow cytometric analysis of DNA content, activation antigen expression, and T cell subset proliferation in the human mixed lymphocyte reaction

This study provides direct correlation via dual parameter flow cytometry (simultaneous assessment of immunofluorescence and DNA content) between mixed lymphocyte reaction (MLR) responder cell entry into the S/G2/M phases of the cell cycle with the kinetics of expression of two activation-associated cell surface proteins, Tac (IL 2 receptor) and 4F2 (unknown metabolic function). A small population of activated cells was identifiable by expression of both Tac and 4F2 antigens before peak DNA synthesis in the MLR. This population of activation antigen-positive cells expanded linearly in size from days 3 to 7 of culture. Treatment of immature MLR cultures with anti-4F2 Mab and complement (C) before DNA synthesis (treatment on day 3, peak DNA synthesis on days 5 to 6) resulted in blunted proliferation and activation antigen expression when the same culture was analyzed after maturation on day 6, indicating that the activated population had been previously detected and removed by anti-4F2 Mab + C. The 4F2 antigen was expressed on a greater percentage of cells in the MLR at all times (days 3 to 9) than was Tac, was present on virtually all S/G2/M phase responder cells, and a large fraction of cells remained intensely 4F2+ subsequent to peak DNA synthesis. In contrast, after initially preceding responder cell entry into the S phase of the cell cycle, the kinetics of Tac antigen expression closely paralleled the kinetics of responder cell proliferation. A subpopulation of cycling responder cells was noted in all MLR cultures studied that expressed Tac antigen weakly or not at all. Cells within both T4 and T8 cell subsets proliferate with similar kinetics in response to alloantigen. The possibility that activation antigens can be utilized to study effector cell generation in the MLR and that this flow cytometric technique may be utilized to analyze the response to various alloantigens is discussed.     

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture.     

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Dual-parameter flow cytometric analysis of an early lymphocyte activation antigen (CK226) and DNA content

This study provides a direct correlation, via dual-parameter flow cytometric analysis (simultaneous assessment of surface immunofluorescence and DNA content), between activated T-cell entry into the S/G2/M phases of the cell cycle and the kinetics of expression of a novel T-cell activation antigen, termed CK226. This molecule was identified by the specific monoclonal antibody on the leukaemic T-cell line CEM/K, and it was expressed by 8-30% of resting peripheral blood lymphocytes and the majority of monocytes and granulocytes. A large fraction of activated lymphocytes acquired the CK226 antigen before DNA synthesis. Moreover, this molecule was expressed on virtually all G0/G1 and S/G2/M phase cells by day 2 after phytohaemagglutinin (PHA) activation and at day 6 after stimulation in a mixed lymphocyte culture. The time course of expression of other known activation antigens, such as Tac and transferrin receptor, was comparable to that of CK226. Based on the relationships between CK226 expression on cycling cells and the stimulatory effects of the specific monoclonal antibody, we conclude that CK226 should be considered an early activation antigen, which defines a new pathway of T-cell activation.     

Overproduction of Rb protein after the G1/S boundary causes G2 arrest.

The Rb protein is known to exert its activity at decision points in the G1 phase of the cell cycle. To investigate whether it may also play some role(s) at later points in the cell cycle, we used a system of rapid inducible gene amplification to conditionally overexpress Rb protein during G2 phase. A cell line expressing a temperature-sensitive simian virus 40 large T antigen (T-Ag) was stably transfected with plasmids containing the Rb cDNA linked to the simian virus 40 origin of replication: pRB-wt, pRB-fs, and pRB-Dra, carrying wild-type murine Rb cDNA, a frameshift mutation close to the beginning of the Rb coding region, and a single-amino-acid deletion in the E1A/T-Ag binding pocket, respectively. Numerous independent cell lines were isolated at the nonpermissive temperature; cell lines displaying a high level of episomal amplification of an intact Rb expression cassette following shiftdown to the permissive temperature were chosen for further analysis. Plasmid pRB-fs did not express detectable Rb antigen, while pRB-Dra expressed full-length Rb protein. The Dra mutation has previously been shown to abrogate phosphorylation as well as T-Ag binding. Fluorescence-activated cell sorting (FACS) analysis revealed that cultures induced to overexpress either wild-type or Dra mutant Rb proteins were significantly enriched for cells with a G2 DNA content. Cultures that amplified pRB-fs or rearranged pRB-wt and did not express Rb protein had normal cell cycle profiles. Double-label FACS analysis showed that cells overexpressing Rb or Rb-Dra proteins were uniformly accumulating in G2, whereas cells expressing endogenous levels of Rb were found throughout the cell cycle. These results indicate that Rb protein is interacting with some component(s) of the cell cycle-regulatory machinery during G2 phase.     

Expression of Epstein-Barr viral capsid, complement fixing, and nuclear antigens in stationary and exponential phase cultures

Three continuous lymphoblastoid cell lines, 2 productive of nucleocapsids and 1 nonproductive line, were studied for their content of Epstein-Barr viral (EBV) antigens during transition from stationary to logarithmic phase growth. As a preliminary step, viable cells were separated from degenerating ones in discontinuous gradients of serum albumin. Viral capsid antigens were found in both living and dead cells of the 2 producer lines; however, complement fixing (CF) antigens and nuclear antigen were detected only in viable cell subpopulations. The content of antigen detectable in extracts of viable cells by complement fixation remained constant in replicating and resting cultures; further, all viable cells of the 3 lines demonstrated intranuclear antigen by anticomplement immunofluorescence in all stages of growth. In contrast, the proportion of cells with viral capsid antigen in the producer lines increased 7- to 24-fold following entry of resting populations into the phase of exponential growth.The results suggest that expression of viral capsid antigens is discontinuous and is initiated in response to events in log phase, possibly DNA synthesis or mitosis. Expression of the complement fixing and nuclear antigens in continuous in viable cells. These findings emphasize the intimate relationship of the CF and nuclear antigen to the transformed state and suggest that study of this antigen complex will shed light on the mechanisms of lymphocyte transformation by EBV.     

Estimation of kinetic cell-cycle-related gene expression in G1 and G2 phases from immunofluorescence flow cytometry data

BACKGROUND: Flow cytometry of immunofluorescence and DNA content provides measures of cell-cycle-related gene expression (protein and/or epitope levels) for asynchronously growing cells. From these data, time-related expression through S phase can be directly measured. However, for G1, G2, and M phases, this information is unavailable. We present an objective method to model G1 and G2 kinetic expression from an estimate of a minimum biological unit of positive immunofluorescence derived from the distribution of specific immunofluorescence of mitotic cells. METHODS: DU 145 cells were stained for DNA, cyclin B1, and a mitotic marker (p105) and analyzed by flow cytometry. The cyclin B1 immunofluorescence (B1) distribution of p105-positive cells was used to model the B1 distribution of G2 and G1 cells. The G1/S and S/G2 interface measurements were used to calculate expression in S phase and test the validity of the approach. RESULTS: B1 at S/G2 closely matched the earliest modeled estimate of B1 in G2. B1 increased linearly through G1 and S but exponentially through G2; mitotic levels were equivalent to the highest G2 levels. G1 modeling of B1 was less certain than that of G2 due to low levels of expression but demonstrated general feasibility. CONCLUSIONS: By this method, the upper and lower bounds of cyclin B1 expression could be estimated and kinetic expression through G1, G2, and M modeled. Together with direct measurements in S phase, expression of B1 throughout the entire cell cycle of DU 145 cells could be modeled. The method should be generally applicable given model-specific assumptions.     

外阴硬化性苔癣组织P53、PCNA表达、DNA含量及与细胞增殖的关系

探讨外阴硬化性苔癣组织中的 P5 3、PCNA表达 ,DNA含量与细胞增殖的关系。免疫组化方法测定 2 0例外阴硬化性苔癣组织和 10例正常外阴皮肤中 P5 3、 PCNA蛋白表达 ;图像分析技术检测两组基底层细胞核形态及 DNA含量。结果显示 ,外阴硬化苔癣组 P5 3阳性表达率为 40 % ,与正常皮肤比较 P<0 .0 5 ,PCNA阳性表达率为 70 % ,与正常皮肤比较 P>0 .0 5 ,阳性表达主要分布于棘层、颗粒层 ;基底细胞核显著变小和 DNA含量降低 (P<0 .0 5 )。结果表明外阴硬化性苔癣组织中存在细胞增殖异常     

Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells

The expression and stability of the proliferation-associated nuclear antigen detected by Ki-67 antibody have been investigated in human promyelocytic leukaemic HL-60 cells in relation to their progression through the cell cycle. Expression of this antigen was minimal in late G1 and early S phase cells. The antigen accumulated in the cells predominantly during S phase, and its rate of increase per cell accelerated during the second half of this phase. The accumulation of Ki-67 antigen during S exceeded the increase in DNA content, and thus the Ki-67/DNA ratio rose 80% from late G1 to G2 + M. This antigen rapidly disappeared from post-mitotic cells. The half-life of this protein estimated in post-mitotic cells during stathmokinesis induced by vinblastine appeared to be shorter than 1 h. This rapid turnover should be compared with the relatively long (6-8 h) duration of G1 of the studied cells. In cells in which de novo protein synthesis was inhibited by 0.1 microgram/ml cycloheximide, the half-life of the Ki-67 antigen was also found to be about 1 h regardless of the cell position in the cell cycle. Thus, the data suggest that variations in the level of this protein during the cell cycle are a consequence of its different synthesis rate rather than phase-specific changes in the rate of its degradation. Because the late G1 and very early S phase cells express the antigen at levels only slightly above background, it is possible that, when using Ki-67 antibody as a marker of the cell growth fraction, some late G1 cells can be erroneously classified as non-cycling cells.     

TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells)

A continuous cell line (YT cells) with inducible receptor for T cell growth factor (TCGF)/interleukin 2 (IL 2) was established from a 15-yr-old boy with acute lymphoblastic lymphoma and thymoma. YT cells were tetraploid, having 4q+ chromosomal markers, and proliferated continuously in vitro without conditioned medium (CM) or IL 2. They were weakly positive for OKT9, OKT11, and Tac antigen (Ag), a determinant closely associated with the receptor for IL 2 (IL 2-R), and were negative for OKT1, OKT3, OKT4, and OKT8 Ag. YT cells also expressed HNK-1 Ag and Fc receptors for IgG, which are expressed on natural killer (NK) cells. They retained a killing activity against human cell lines, including K562 (myeloid), T, and B cell lines. Unlike Tac Ag/IL 2-R(+) cell lines derived from adult T cell leukemia (ATL), YT cells were negative for HTLV, as proved by Southern blotting with cDNA for viral DNA. The expression of Tac Ag was markedly enhanced in 18 hr, when YT cells were incubated with CM from PHA-stimulated peripheral blood leukocytes (PBL) or spleen cells, as determined by immunofluorescence by using flow cytometry and binding assay with 125I-anti-Tac antibody (Ab). The binding study with 125I-labeled recombinant IL 2 showed 3.2 X 10(4) IL 2 receptor sites on YT cells precultured with CM. PHA-P and Con A neither agglutinate nor enhance the expression of IL 2-R/Tac antigen on these non-T cell line cells. Furthermore, neither recombinant IL 2 nor gamma-interferon could induce IL 2-R on YT cells, suggesting the presence of a unique IL 2-R inducing factor in PBL or spleen CM. Unlike Tac Ag on HTLV(+), ATL-derived cell lines (Hut-102, MT-1, ATL-2), the expression of Tac Ag on YT cells was down-regulated by anti-Tac Ab. The induction of Tac Ag/IL 2-R on YT cells seemed specific, because the enhancement of Tac Ag expression was not associated with that of Ia Ag and T9/transferrin receptor.     

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability.

BACKGROUND: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. MATERIALS AND METHODS: Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. RESULTS: Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD-negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. CONCLUSIONS: The results show that cells stained with FITC-labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions.     

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 (G_1a and late G₁ (G_lb) while others may come out of mitosis committed to go through the following cycle by directly entering late G₁ compartment.     

Simultaneous analysis of DNA content and surface antigens in human bone marrow

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell-surface antigens and DNA content on bone marrow cells while preserving their light-scatter properties. Proliferation in a population defined by light-scatter and surface-antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA ("proliferation index"). Viable, low-density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV-excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light-scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest "proliferation index" detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

Therapeutic efficacy of natural dipeptide carnosine against human cervical carcinoma cells

Natural substances have been attracted several researchers in the recent years, because of its potential antioxidant, anti‐inflammatory and anti‐cancer properties. We have investigated the effect of carnosine on cell viability, apoptosis, DNA damage, reactive oxygen species (ROS) and caspase 3 enzyme expression in human cervical carcinoma and Madin‐Darby Kidney Cells (MDCK) cells . Carnosine inhibited cancer cell growth up to 23%. ROS level was increased up to 30 and 31% in MDCK and HeLa cells respectively. Tunnel assay showed 42 and 14% of positive apoptotic cells in cancer and normal cells respectively. The alteration in mitochondrial and nuclear morphology was determined. The extended lace‐like network of normal mitochondria found in control cells. Carnosine treatment significantly altered the mitochondrial morphology of normal cervical carcinoma cell. Mitochondria were condensed clump structures in carnosine treated cancer cells. Carnosine reduced the number of colonies of cervical carcinoma cells. Caspase 3 expression was corresponded to the appearance of immunofluorescence in the cytoplasm. Caspase 3 expression was gradually increased in cervical carcinoma cells. In Silico, docking study was performed to recognize the binding activity of carnosine against a subunit of the caspase 3 , and carnosine was able to bind to the drug binding pocket of caspase 3. The glide energy is ?5.2 kcal/mol, suggesting the high binding affinity of carnosine to caspase 3. Taking all these data together, the natural dipeptide L‐carnosine could be a suitable antiproliferative agent in cervical carcinoma cells. Copyright © 2016 John Wiley & Sons, Ltd.     

Human cytomegalovirus infection inhibits G1/S transition.

Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G1/S (hydroxyurea and mimosine) and G2/M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G0) cells, the cell cycle did not progress beyond the G1/S border even after serum stimulation. Proteins which normally indicate G1/S transition (proliferating cell nuclear antigen [PCNA]) or G2/M transition (cyclin B1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B1 was induced in infected cells which exhibited a G1/S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.     

The relationship between thrombomodulin expression and cell cycle stages in cultured rabbit endothelial cells

Cultured rabbit endothelial cells have significant but variable amounts of thrombomodulin (TM), both on their surface as well as inside the cell. To determine if variations in TM antigen is cell cycle related, cells with very high levels of TM antigen were identified and staged according to the intracellular distribution and relative amounts of the antigen, using immunofluorescence techniques. After staging, the nuclear DNA content of each of these cells was determined by measuring the propidium iodide (PI) fluorescence intensity cytophotometrically. Stages 1, 2, and 3, which exhibited TM immunofluorescence in the golgi area, clustered to the G1 phase of the cell cycle. Cells without discernible golgi fluorescence (stages 4 and 5) but with variable amounts of cytoplasmic and surface fluorescence appeared to have little or no relationship to the cell cycle.     

A unique antigen on mature B cells defined by a monoclonal antibody

A novel 42,000 dalton antigen (MB-1) expressed by mature human B cells in blood and tonsil was identified and characterized by utilizing a hybridoma monoclonal antibody. A comparison of MB-1 with other known B cell antigens suggests that the MB-1 antigen has not been previously identified. From one-and two-color immunofluorescence studies, it appears that the MB-1 antigen is found on all normal immunoglobulin (Ig)-expressing cells, but not on T cells, thymocytes, granulocytes, or platelets. Studies of malignant B cell tumors reveal that the antigen is expressed by virtually all Ig-expressing B cell tumors but only 10% of SIg- B-lineage leukemias. Data from these studies suggest that the MB-1 antigen is expressed late in B cell ontogeny before the expression of SIg.