The cell cycle associated change of the Ki-67 reactive nuclear antigen expression

The change of human nuclear antigen expression in proliferating cells recognized by a monoclonal antibody, Ki-67, during the cell cycle was investigated in HeLa S3 cells using a bivariate-flow-cytometric analysis. The antigen was immunocytochemically stained with FITC, and DNA was stained with propidium iodide (Pl). The expression of the antigen increased with cell-cycle progression, especially in the latter half of S-phase and reached a maximum at G2M-phase, although its content varied greatly from cell to cell. The cells in which DNA synthesis was inhibited by treatment with hydroxyurea increased markedly in the antigen expression (as compared to untreated cells). Treatment with adriamycin also elevated the antigen content. After digestion with DNase I, but not after RNase treatment, FITC fluorescence from the antigen disappeared. These results suggest that the Ki-67 antigen is bound to DNA and its expression does not depend on DNA replication. Although the biological implications of the antigen remain unresolved, the antigen may be considered to be essential for maintaining the proliferating state of cells.     

Cell cycle regulated expression of nucleolar antigen P120 in normal and transformed human fibroblasts

Normal and SV40 virus-transformed WI-38 human lung fibroblasts were serum starved and refed, or synchronized by double thymidine block and released from the block. At different time points in the cell cycle, steady state levels of P120 mRNA and P120 protein content of the cells were determined by densitometric scans of Northern and Western blots. At the same time points, [³H]thymidine uptake was measured and flow cytometric analysis performed for DNA content and P120 antigen staining. Levels of P120 protein and P120 mRNA were approximately 4 times greater in non-synchronous, exponentially growing transformed cells than in similarly growing normal cells. Early G₁-cells, synchronized either with serum deprivation or with metabolic block, contained only a trace amount of P120 protein and mRNA. The P120 gene was transcribed early in G₁ and P120 protein synthesis initiated in middle G₁. A dramatic increase of P120 protein level occurred in S-phase with a corresponding mRNA peak preceding the P120 protein peak. These results indicate that P120 is overexpressed in transformed WI-38 cells and that P120 is temporally regulated during the cell cycle of both transformed and normal fibroblasts. The dramatic increase in P120 protein expression at the G₁ to S boundary suggests that P120 may play a role in the regulation of cell cycle and increased nucleolar activity that is associated with cell proliferation. © 1993 Wiley-Liss, Inc.     

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.     

Middle T antigen as primary inducer of full expression of the phenotype of transformation by polyoma virus.

A large number of polyoma virus-transformed cells of rat, mouse, and hamster origin were examined for presence of T-antigen species. The results showed that all lines of cells contained middle and small T antigens, but not all contained a full-sized large T antigen, in some cell lines large T antigen was absent, whereas in others it was present as truncated forms lacking various lengths of the carboxy-terminal part of the protein. Cells transformed by the new viable deletion mutants of polyoma virus, dl-8 and dl-23, formed larger and smaller colonies or foci, respectively, when they were suspended in semisolid medium or plated as monolayers together with untransformed cells on a plastic surface. The deletions in the DNA of these mutants resulted in the shortening of the large and middle T antigens simultaneously without affecting the size of the small T antigen. Variation of large T-related proteins in dl-8 and dl-23-transformed cells seemed to be the same as that observed in wild-type-transformed cells. Regardless of the amount and size of large T-related protein in mutant-transformed cells, the phenotype of the cells was entirely dependent on the mutant used. The results suggest that (i) persistence of large T antigen is not universally required for the maintenance of the transformation phenotype, (ii) small T antigen alone may not be sufficient for inducing the full expression of the transformation phenotype, and (iii) middle T antigen is implicated as being primarily responsible for the full expression of the phenotype of transformation. The results also provide the evidence that the carboxy-terminal region of middle T antigen and a part of large T antigen are encoded in the genome in the same DNA segment around map units 88 to 94 in different reading frames.     

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.     

Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells.

Primary or continuous lines of mouse cells (3T3) are nonpermissive for simian virus 40 (SV40). Abortively infected cells synthesize tumor antigen (T antigen but not viral DNA and virus capsid protein (V antigen). V antigen, however, was obtained when SV40 DNA was injected into 3T3 cells. This late gene expression also appears to be correlated with the quantity of injected DNA molecules per 3T3 cell. T antigen formation can be detected after microinjection of only 1 to 2 DNA molecules, but the intensity of intranuclear T antigen fluorescence is significantly brighter with injection of higher concentrations of viral DNA. In permissive cells (TC7), early and late SV40 gene expression is directly related to the number of injected molecules. Microinjection of 1DNA molecule induced T and V antigen formation with the same efficiency as microinjection of 2,000 to 4,000 molecules. The question of weather late SV40 gene expression is directly related to the quantity of an early virus-specific product was approached by microinjection of early SV40 complementary RNA together with small amounts of viral DNA. V antigen was obtained in a high proportion of recipient 3T3 cells at conditions where microinjection of viral DNA alone induced T but not V antigen synthesis.     

Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA

Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression and characterization of a parasite-specific antigen on macrophages after infection withLeishmania donovani

A rabbit polyclonal antibody to crude soluble antigen ofLeishmania donovani promastigotes recognized a determinant expressed on the surface membrane of mouse peritoneal macrophages and human monocyte derived macrophages infectedin vitro. The determinant was recognized on infected macrophage surface only when F (ab)₂ fragments of anti-leishmanial antiserum was employed in immunofluorescence. F(ab)₂ fragments of human patient sera also could recognize the determinant. The expression of this antigen was not stage-specific for the parasite. Immunochemical analyses revealed this antigen to be of 51 kDa protein. Specific leaching of membrane proteins by trypsin showed three bands of expressed antigens of 26, 11 and 10 kDa, which in all likelihood might be arising from the 51 kDa antigen. The antigen was not expressed until 12 h of post infection, reached a maximum level at 24 h and thereafter attained a steady state level as studied upto 96 h of post infection. This typc of antigen might have a great potential in immunodiagnostics and site-specific drug targeting.     

Control of cell cycle entry and progression in mitogen-stimulated human B lymphocytes

Tonsillar B lymphocytes were stimulated to proliferate by the mitogenic combination of phorbol dibutyrate and ionomycin. Progression through the cell cycle was monitored by measurements of cellular DNA and RNA content using flow cytometry. Changes in surface expression of class II MHC antigens and CD20 antigen were also monitored as early parameters of B lymphocyte activation and cell cycle progression. The results showed that about 60% of the population synchronously entered and progressed through the cell cycle. The transition from the resting state, signaled by increased RNA content, occurred about 12 to 24 hr after stimulation; S phase entry occurred at about 36 hr. Small, variable populations of cells appeared to be unresponsive to the stimuli, either because they were “preactivated” before in vitro stimulation or were already dying. The kinetics of appearance and accumulation of several cell cycle regulated/regulatory proteins were followed by immunoblotting. The proliferating cell nuclear antigen (PCNA) cyclin A and p33^cdk2 proteins were either absent or present in very low amounts in resting cells and first became detectable in increased amount beginning at about 24 hr after stimulation; increased p34^cdc2 protein was not detected until about 36 hr. Increased cellular content and phosphorylation of the p110^Rb protein was already obvious by 24 hr after stimulation. The effects of several immunosuppressive agents were examined using purified B cells. Both cyclosporin A and an FK506 analogue were shown to inhibit proliferation of B lymphocytes, at the low doses also inhibitory to T cells. © 1995 Wiley-Liss, Inc.     

Macronuclear structure of the G surface antigen gene of Paramecium primaurelia and direct expression of its repeated epitopes in Escherichia coli.

The gene encoding the G surface antigen of Paramecium primaurelia was cloned from a macronuclear DNA library by a screening procedure involving differential hybridization with cDNA probes synthesized from polyadenylated RNAs of cells expressing one of two alternate antigens. S1 mapping experiments and sequencing of the cloned DNA and the mRNA showed that the cloned gene corresponded to the high-molecular-weight mRNA that had been indirectly identified as that of the G surface antigen. Because the genetic code of Paramecium spp. is different from the "universal" code, this mRNA cannot be correctly translated in vitro; direct proof that it encoded the antigenic determinants of this protein was therefore obtained through expression of fragments of the coding sequence in Escherichia coli by using the expression vector lambda gt11. Studies on the structure of this gene revealed that the central part of the coding sequence contained at least five tandem repeats of 222 base pairs, encoding immunogenic domains of the protein. We also showed that, like other surface antigen genes of trypanosomes and paramecia, this gene lay next to a chromosome end and that no rearrangement of its immediate genomic environment was associated with its expression.     

Revealing the potential of DNA-based vaccination: lessons learned from the hepatitis B virus surface antigen

DNA-based vaccination is a novel technique to efficiently stimulate humoral (antibody) and cellular (T cell) immune responses to protein antigens. In DNA-based vaccination, immunogenic proteins are expressed in in vivo transfected cells of the vaccine recipients in their native conformation with correct posttranslational modifications from antigen-encoding expression plasmid DNA. This ensures the integrity of antibody-defined epitopes and supports the generation of protective (neutralizing) antibody titers. Plasmid DNA vaccination is furthermore an exceptionally potent strategy to stimulate CD8+ cytotoxic T lymphocyte (CTL) responses because antigenic peptides are efficiently generated by endogenous processing of intracellular protein antigens. These key features make DNA-based immunization an attractive strategy for prophylactic and therapeutic vaccination against extra- and intracellular pathogens. In this brief review, we summarize the current state of expression vector design, DNA delivery strategies, priming immune responses to intracellular or secreted antigens by DNA vaccines and unique advantages of DNA- versus recombinant protein-based vaccines using the hepatitis B surface antigen (HBsAg) as a model antigen.     

Hypersensitivity of Ku-Deficient Cells toward the DNA Topoisomerase II Inhibitor ICRF-193 Suggests a Novel Role for Ku Antigen during the G2 and M Phases of the Cell Cycle

Ku antigen is a heterodimer, comprised of 86- and 70-kDa subunits, which binds preferentially to free DNA ends. Ku is associated with a catalytic subunit of 450 kDa in the DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double-strand break (DSB) repair and V(D)J recombination of immunoglobulin and T-cell receptor genes. We now demonstrate that Ku86 (86-kDa subunit)-deficient Chinese hamster cell lines are hypersensitive to ICRF-193, a DNA topoisomerase II inhibitor that does not produce DSB in DNA. Mutant cells were blocked in G₂ at drug doses which had no effect on wild-type cells. Moreover, bypass of this G₂ block by caffeine revealed defective chromosome condensation in Ku86-deficient cells. The hypersensitivity of Ku86-deficient cells toward ICRF-193 was not due to impaired in vitro decatenation activity or altered levels of DNA topoisomerase IIα or -β. Rather, wild-type sensitivity was restored by transfection of a Ku86 expression plasmid into mutant cells. In contrast to cells deficient in the Ku86 subunit of DNA-PK, cells deficient in the catalytic subunit of the enzyme neither accumulated in G₂/M nor displayed defective chromosome condensation at lower doses of ICRF-193 compared to wild-type cells. Our data suggests a novel role for Ku antigen in the G₂ and M phases of the cell cycle, a role that is not related to its role in DNA-PK-dependent DNA repair.     

Cell heterogeneity and subpopulations in solid tumors characterized by simultaneous immunophenotyping and DNA content analysis

BACKGROUND: Heterogeneity in human malignant tumors is a well-described phenomenon and of interest with regard to subpopulations with differences in clonality, metastatic potential, and response to therapy under different treatment regimes. The aim of this study was the simultaneous characterization of surface markers and DNA content of solid tumors to identify tumor cell subpopulations and to study the association between the expression of antigens and DNA content. METHODS: In the present study, six different malignant tumors grown as xenografts in nude mice were characterized by five-parameter flow cytometry. Immunophenotyping was performed using a variety of direct fluorescence-conjugated antibodies. In all cases, simultaneous detection of DNA content was done after staining with 7-aminoactinomycin D. RESULTS: Tumor cells were characterized by light scatter properties, antigen expression, and DNA content. Tumor cell heterogeneity, subpopulations, and DNA content-dependent antigen expression were identified. CONCLUSIONS: This method offers the possibility of characterizing solid tumors according to their immunophenotype and DNA content. The results obtained can be used to identify changes in immunophenotypic and DNA profiles of tumor cell populations before and after therapy and might be useful to define parameters predictive for response to therapy.     

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.     

Analysis of T cell receptor Vβ gene usage during the course of disease in patients with chronic hepatitis B

The T cell receptor (TCR) is a heterodimeric molecule expressed on the surface of T cells and recognizes foreign peptides presented by the major histocompatibility complex on the surface of antigen-presenting cells or virusinfected cells. Analysis of TCR usage by T cells which recognize hepatitis B virus (HBV) provides further insight into the participation of T cell populations during the course of disease. In this study, we examined the T-cell-proliferative response and the TCR V gene usage of peripheral blood mononuclear cells in 3 patients with clinical evidence typical of chronic hepatitis B. All 3 patients had significant T-cell proliferative responses against HBV core antigen (HBcAg) during the remission stage, while no responses were detected during the acute exacerbation stage. In addition, the TCR V₇ gene was utilized more frequently in T cells recognizing HBcAg during remission, while TCR V₁ and V₂ were utilized at a higher percentage during acute exacerbation. On the contrary, the T cell proliferative response against HBV surface antigen was undetectable and no specific V gene was utilized more frequently by all 3 patients, regardless of disease state. Our longitudinal studies, although based on a small sample of patients, demonstrate that the population of HBcAg-activated T cells alters during the course of disease in chronic hepatitis B patients.     

Prognostic significance of expression of antigens on melanoma cells

Summary The expression of antigens on 33 human melanoma cells obtained directly from surgically excised tumours was investigated by means of antibody-dependent cell-mediated cytotoxic assays. Antisera used in the study were two antisera from human melanoma patients against different tumour-associated antigens on melanoma cells and antisera against carcinoembryonic antigen (CEA) and ₂ microglobulin (₂M). Considerable heterogeneity was observed in the expression of both melanoma-associated and non-melanoma antigens on melanoma cells from 33 different patients.Patients whose tumours were reactive with the melanoma-associated antiserum (CHI) had a significantly longer remission period to stage 2 melanoma. The period to development of stage 3 melanoma also appeared longer, but this was not statistically significant with the number of patients available for study. The expression of CEA, ₂M, and the tumour-associated antigen TIN was not significantly related to the recurrence-free interval. There appeared to be a reciprocal expression of the two melanoma-associated antigens, and patients with tumours expressing CHI but not TIN had a significantly longer recurrence-free interval than patients whose tumours had the opposite antigenic pattern.In the limited number of patients available for study the expression of the antigen CHI did not appear related to thickness of the primary tumour or to immune response of the patients to melanoma cells in leucocyte-dependent antibody and natural killer cell assays. Although the nature of the association between expression of this antigen and longer remission-free period is unknown these results suggest that the expression of certain melanoma antigens on the cell surface may be an important additional variable which has prognostic and therapeutic importance.     

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     

Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G₁, most virus transformed or tumor cells are unaffected in G₁ but arrest in G₂ phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nucleolar p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 μM SSP.MOLT-4 cells did not arrest in G₁ or G₂ phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G₁ phase at higher DNA ploidy (tetraploidy; G_1T), and then progressed through S_T (rereplication) into G_2T and M_T. The rates of entrance to G₂ and G_2T were essentially identical, indicating that the rates of cell progression through S and S_T as well as through G₂ and G_2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 μM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis. © 1994 Wiley-Liss, Inc.