Duration of the cell cycle phases in mutants for the tumor suppressor <Emphasis Type="Italic">Merlin</Emphasis> in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The cell cycle duration was estimated in Drosophila melanogaster mutants for the tumor suppressor Merlin with the use of different approaches. Experiments on induction of mosaic clones in tissues of the larval wing imaginal disc showed that the cell cycle in mutant discs is shorter than that in control. Flow fluorescence cytometry revealed no differences between mutant and normal animals in the relative duration of the cell cycle phases, which suggests proportional shortening of the cell cycle phases. The study with pulselabeled mitoses confirmed these results and showed that the length of the cell cycle is 7 h (S phase duration 3 h) in control individuals and 5 h (S phase duration 2 h) in Merlin gene mutants.     

The classical nuclear localization signal receptor, importin-alpha, is required for efficient transition through the G1/S stage of the cell cycle in Saccharomyces cerevisiae

There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-alpha/Srp1, to the G(2)/M transition of the cell cycle. Here, we utilize two engineered mutants of importin-alpha/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G(1) to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest G(1)/S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in G(2)/M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-alpha mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the G(1)/S cell cycle transition.     

Polyomavirus small T antigen controls viral chromatin modifications through effects on kinetics of virus growth and cell cycle progression

Minichromosomes of wild-type polyomavirus were previously shown to be highly acetylated on histones H3 and H4 compared either to bulk cell chromatin or to viral chromatin of nontransforming hr-t mutants, which are defective in both the small T and middle T antigens. A series of site-directed virus mutants have been used along with antibodies to sites of histone modifications to further investigate the state of viral chromatin and its dependence on the T antigens. Small T but not middle T was important in hyperacetylation at major sites in H3 and H4. Mutants blocked in middle T signaling pathways but encoding normal small T showed a hyperacetylated pattern similar to that of wild-type virus. The hyperacetylation defect of hr-t mutant NG59 was partially complemented by growth of the mutant in cells expressing wild-type small T. In contrast to the hypoacetylated state of NG59, NG59 minichromosomes were hypermethylated at specific lysines in H3 and also showed a higher level of phosphorylation at H3ser10, a modification associated with the late G(2) and M phases of the cell cycle. Comparisons of virus growth kinetics and cell cycle progression in wild-type- and NG59-infected cells showed a correlation between the phase of the cell cycle at which virus assembly occurred and histone modifications in the progeny virus. Replication and assembly of wild-type virus were completed largely during S phase. Growth of NG59 was delayed by about 12 h with assembly occurring predominantly in G(2). These results suggest that small T affects modifications of viral chromatin by altering the temporal coordination of virus growth and the cell cycle.     

Characteristics of the cell cycle of matrix cells in the mouse embryo during histogenesis of telencephalon

The cell cycle of matrix cells in the telencephalon of the mouse embryo at different stages at day 10, 13, and 17 of gestation was investigated by means of ³H-thymidine autoradiography.The cell cycle time of matrix cells in the day 10 group was found to be 7.0 h, and lengthened linearly with embryonic age. The cell cycle times of day 13 and 17 groups were 15.5 and 26.0 h, respectively.The duration of G1 and S phases also lengthened linearly with embryonic age. The durations of G1 phase were 0.1, 6.8, and 13.8 h, for day 10, 13, and 17 groups, respectively, and those of S phase were 5.1, 6.9, and 10.4 h, for day 10, 13, and 17 groups, respectively. On the other hand, the durations of both G2 and M phases remained unchanged and these were 1.0 and 0.8 h, respectively, throughout the embryonic stages.It was a characteristic of the alteration of the cell cycle of the telencephalon during mouse embryonic life that not only G1 but also S phases lengthened linearly with embryonic age and both G2 and M phases remained constant.     

Modulation of antioxidant enzymes,reactive oxygen species,and glutathione levels in manganese superoxide dismutase-overexpressing NIH/3T3 fibroblasts during the cell cycle

NIH/3T3 mouse embryo fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD). Previous studies showed characteristic unique AE profiles in nonsynchronous populations of parental, control plasmid-transfected, and MnSOD-overexpressing NIH/3T3 cell lines. However, the present study showed that during S and M phases of the cell cycle, antioxidant enzyme (AE) levels were altered in MnSOD-overexpressing cell lines towards levels in S and M phases of parental and control plasmid-transfected cells. Because of the demonstration that MnSOD overexpression inhibits cell growth in both nonmalignant and malignant cells, the present study was designed to measure AEs, reactive oxygen species (ROS), and glutathione levels in various phases of the cell cycle in both parental NIH/3T3 cells and NIH/3T3 cells overexpressing MnSOD, to try to determine whether AEs, ROS, and glutathione levels could have a possible regulatory role in cell cycle progression. In all cell lines studied, ROS levels were lower in M than S phase of the cell cycle. Total glutathione and glutathione disulfide levels were greatly increased during the M phase of the cell cycle compared with quiescence and S phase in all cell lines studied. This suggests that oxidative stress exists in M phase of the cell cycle with total glutathione levels increased to decrease oxidative stress. Analysis of MnSOD-overexpressing cell clones showed a correlation of decreased cell growth with an increase in ROS in S phase of the cell cycle and a decrease in glutathione in mitosis. The data strongly suggest that specific levels of cell redox state are necessary for cells to successfully progress through the various phases of the cell cycle. J. Cell. Physiol. 177:148–160, 1998. © 1998 Wiley-Liss, Inc.     

Cell Cycle Arrest of Cdc Mutants and Specificity of the Rad9 Checkpoint

In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G(2) phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G(2) phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G(2) phase.     

Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae

The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S phase occupying the middle half of the cell cycle. In each growth condition, start appeared to correspond to the G1 phase/S phase boundary. Bud emergence did not occur until mid S phase. These results show that the rate of transit through all phases of the cell cycle can vary considerably when cell cycle length changes. When cells growing at different rates were arrested in G1, the following synchronous S phase were of the duration expected from the length of S in each asynchronous population. Cells transferred from a poor nitrogen source to a good one after arrest in G1 went through the subsequent S phase at a rate characteristic of the better medium, indicating that cells are not committed in G1 to an S phase of a particular duration.     

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry

Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G1, S and G2 + M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about two-fold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle (Tc) was 15.3 h for PB-3 cells and 12.4 h for PB-1 cells. Shortening in Tc for the transformed cells was due to a decrease of nearly 30% in mean duration of the G1 phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G2 + M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G1 phase of the cell cycle.     

Estimation of circadian variations in cell cycle phase durations in murine epidermal basal cells

Circadian variations in the proliferative activity of squamous epithelia are well known. However, circadian variations in the duration of the various cell cycle phases (S, G2 and mitosis) have been disputed. The percent labelled mitoses method, which is traditionally used to obtain duration of cell cycle phases, is poorly suited for identification of circadian variations. Therefore methods combining changes in compartment size (cell cycle phase) and cellular flux through the compartments have been used. Three different methods using such data are presented. These incorporate various simplifying assumptions that cause methodological errors. Limits for use of the different methods are indicated. The use of all three methods gives comparable and pronounced circadian variations in the duration of S and G2 phase. These results are also compatible with circadian variations in the mitotic duration, but they may also represent artefacts due to sensitivity to model errors.     

Studies on the Growth in Culture of Plant Cells: XVII. ANALYSIS OF THE CELL CYCLE OF ASYNCHRONOUSLY DIVIDING ACER PSEUDOPLATANVS L. CELLS IN SUSPENSION CULTURE

Three methods of cell cycle analysis, involving the use of tritiatedthymidine, have been applied to asynchronously dividing suspensioncultures of sycamore. Conditions for an effective chase of unlabelledthymidine were established from a study of the kinetics of entryand incorporation of tritiated thymidine into the cells. Thelevels of thymidine used did not affect the rate of cell divisionor the duration of the phases of the cell cycle. The analyses of the cell cycle based upon pulse labelling, continuouslabelling, and a combination of densitometry and autoradiographywere in good agreement and showed that the phases S (mean 7.0h), G2 (mean 8.5 h) and mitosis (mean 3.0 h), were of relativelyconstant duration, whereas G1 was of variable duration. No relation between nuclear DNA content and mitotic-cycle timeor the duration of S-phase could be inferred from the data presented.     

Simian virus 40 large T-antigen expression decreases the G1 and increases the G2 + M cell cycle phase durations in exponentially growing cells.

The effect of simian virus 40 large T-antigen (Tag) expression on the cell cycle of exponentially growing, established, mouse NIH 3T3 fibroblasts was examined by using a sensitive flow cytometric assay to analyze nonselected cells immediately after infection with a Tag-encoding recombinant retrovirus. Tag expression resulted in reduced percentages of G1-phase cells and increased percentages of S- and G2 + M-phase cells compared with cell populations infected with a control virus not encoding the Tag gene. Cell cycle-blocking drugs were used to examine the exit rate for each of the cell cycle phases, G1, S, and G2 + M, for Tag-expressing and Tag-nonexpressing cells growing in the same cell culture dish. As a result of Tag expression, the duration of the G1 phase was decreased (average G1-phase exit duration decreased by 18%) and the duration of the G2 + M phase was increased (average G2 + M exit duration increased by 29%). The duration of S phase was unaffected by Tag expression.     

Hyperthermia responses in cell lines with normal and deficient DNA repairs systems

Earlier data showed that cultured cells are more hyperthermia sensitive in S phase of the cell cycle. In part this sensitivity may be related to the disruption of DNA repair/processing activity in S phase. It is well known that many components of DNA repair systems are in fact involved in DNA replication/processing. For this study we set out to evaluate a wide range of DNA repair mutants to determine their thermal responses compared to the wild-type cells. Mutants of nucleotide excision repair (NER), homologous recombination repair (HR) and nonhomologous endjoining (NHEJ) were studied. In all cases the mutants were more thermally sensitive than the wild-type counterparts. In addition, studies on thermal tolerance (TT) showed that mutants did not have a smaller TT response than the wild type thus ruling out TT as a cause in the increased sensitivity. Cell cycle analysis showed no significant difference amongst mutant and wild-type cell line pairs and thus effects of differing cell cycle distribution was also ruled out. It is speculated that it may be the involvement of the mutated pathways in DNA replication/progressing that make mutated cells more sensitive than the wild types.     

p21CDKN1A Does Not Interfere with Loading of PCNA at DNA Replication Sites,but Inhibits Subsequent Binding of DNA Polymerase D at the G1/S Phase Transition

The ability of the cyclin-dependent kinase (CDK) inhibitor p21CDKN1A to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G1 phase cell cycle arrest induced by p21GFP occurred also at the G1/S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21PCNA-) or CDK (p21CDK-) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21wtHA and p21CDK-HA, but not p21PCNA-, co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase d (pol d). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol d to PCNA at the G1/S phase transition.     

p21CDKN1A does not interfere with loading of PCNA at DNA replication sites, but inhibits subsequent binding of DNA polymerase delta at the G1/S phase transition

The ability of the cyclin-dependent kinase (CDK) inhibitor p21CDKN1A to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G1 phase cell cycle arrest induced by p21GFP occurred also at the G1/S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21PCNA-) or CDK (p21CDK-) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21(wt)HA and p21CDK-HA, but not p21PCNA-, co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase delta (pol delta). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol delta to PCNA at the G1/S phase transition.     

Isolation by a replica-plating technique of chinese hamster temperature-sensitive cell cycle mutants

Isolation of a wide variety of temperature-sensitive (ts) cell cycle mutants in mammalian cells has previously proved to be a very difficult task. The various procedures used for the isolation of such mutants included a mutant enrichment step based on exposure of the cells to the restrictive temperatures in order to kill the growing wild-type cells with agents that kill DNA-synthesizing cells. Hence, these methods favored the isolation of ts mutants that do not lose viability rapidly at the restrictive temperatures, We have treated cells of the Chinese hamster established cell line E36 with the mutagen ethyl-methane-sulfonate (EMS) and used a replicaplating technique that we developed to screen the ts mutants for growth. This technique enabled us to recover all ts mutants for growth including the ts cell cycle mutants. Screening of the ts cell cycle mutants among the ts mutants for growth was performed by the flow microfluorimetry technique and the premature chromosome condensation technique. Our results show that 1.3% of the survivors of the mutagenic treatment are ts mutants for growth. Six of 84 ts mutants analyzed were found to be ts cell cycle mutants. They include ts mutants arrested in phases G1, S, and G2. Many of the ts mutants for growth including the ts cell cycle mutants arrested in S and G2 lose viability very fast when incubated at the restrictive temperature. As a consequence they could not have been isolated by any method that includes a mutant enrichment step based on the exposure of the cells to the restrictive temperature.     

Male germ line development in Arabidopsis. duo pollen mutants reveal gametophytic regulators of generative cell cycle progression

Male germ line development in flowering plants is initiated with the formation of the generative cell that is the progenitor of the two sperm cells. While structural features of the generative cell are well documented, genetic programs required for generative cell cycle progression are unknown. We describe two novel Arabidopsis (Arabidopsis thaliana) mutants, duo pollen1 (duo1) and duo pollen2 (duo2), in which generative cell division is blocked, resulting in the formation of bicellular pollen grains at anthesis. duo1 and duo2 map to different chromosomes and act gametophytically in a male-specific manner. Both duo mutants progress normally through the first haploid division at pollen mitosis I (PMI) but fail at distinct stages of the generative cell cycle. Mutant generative cells in duo1 pollen fail to enter mitosis at G2-M transition, whereas mutant generative cells in duo2 enter PMII but arrest at prometaphase. In wild-type plants, generative and sperm nuclei enter S phase soon after inception, implying that male gametic cells follow a simple S to M cycle. Mutant generative nuclei in duo1 complete DNA synthesis but bypass PMII and enter an endocycle during pollen maturation. However, mutant generative nuclei in duo2 arrest in prometaphase of PMII with a 2C DNA content. Our results identify two essential gametophytic loci required for progression through different phases of the generative cell cycle, providing the first evidence to our knowledge for genetic regulators of male germ line development in flowering plants.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.     

Analysis of the cell cycle in the root meristem of Allium cepa under the influence of ledakrin

The influence of a polish anticancer drug on the cell cycle using Allium test was studied. Methods of aceto-orcein squash slides, curve of labelled mitoses after 3H-thymidine incubation and cytophotometrics after Feulgen's reaction were employed. Ledakrin acts strongly antimitotically, but it does not block the cell cycle completely. The cytostatic activity of ledakrin results from its action on the interphase. The phases G1 and S are prolonged while M is unchanged after 6h incubation with ledakrin. During postincubation in water without ledakrin it was noted, at the beginning, that the mitotic activity decreases and it is brought about the lengthening of S and G2 phases. The duration of the cell cycle phases returns to the control level during further postincubation. The results of analysis of chromatin aberrations and the micronucleus test point to a mutagenic effect of ledakrin.     

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry

Abstract. Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G¹, S and G₂+ M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about twofold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle ( T _c) was 15-3 h for PB-3 cells and 12-4 h for PB-1 cells. Shortening in T _c for the transformed cells was due to a decrease of nearly 30% in mean duration of the G ₁ phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G₂+ M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G¹ phase of the cell cycle.