Cellular content of ribosomal RNA in relation to the progression and competence signals governing proliferation of 3T3 and SV40-3T3 cells

The method for differential fluorescence staining of cellular RNA and DNA by acridine orange (AO) was optimized for 3T3 and SV40-3T3 cells. Cellular contents of DNA and of ribosomal RNA (rRNA) were determined by dual-channel flow cytometry during cell-density-dependent proliferation and after stimulation of quiescent cells. With increasing density of 3T3 cells, cellular content of rRNA decreases by about 60%, whereas SV40-3T3 cells do not exhibit a significant dependence of rRNA content on cell density. 3T3 cells stimulated early after becoming quiescent resume reaccumulation of rRNA after a delay of only 4 h, whereas cells maintained at quiescence for several days exhibit a delay of about 12 h before a significant rise of rRNA is observed. The extent of rise of cellular rRNA content after different regimens of stimulation of quiescent 3T3 cells does not correlate well with the fraction of cells entering the cell cycle. These and other reported instances of discordance between rRNA content and stimulation into the cell cycle are resolved by showing that of the two signals governing entry into the cell cycle only the progression signal, but not the competence signal is associated with reaccumulation of cellular rRNA. The present results are consistent with the progression function being in essence the achievement of a threshold number of ribosomes per cell, which in conjunction with the competence signal is sufficient for initiation of the cell cycle.     

Level of ribosomal RNA required for stimulation from quiescence increases during cellular aging in vitro of mammalian fibroblasts

We have investigated the relation between cell size in terms of cellular ribosomal RNA (rRNA) content and proliferation of diploid human and rat embryo fibroblasts during their aging in vitro. During phase III of the proliferative lifespan in vitro, cellular rRNA content increases by a factor of nearly 3. For very different regimes of stimulation of quiescent cells, a strict correlation was observed, between the proportion of cells stimulated and cellular rRNA content, resembling a steep threshold curve. During aging in vitro, these characteristic curves exhibit an essentially parallel shift to higher values of cellular rRNA content (to higher 'thresholds'). Upon establishment as a permanent cell line, the relation between proliferation stimulation and cellular rRNA ceases to change with further subculturing. It is suggested that the essence of transformation of fibroblasts with a myc-type of oncogenes is a reduction and stabilizing of the critical rRNA content required for proliferation.     

A cell regulatory agent, CeReS-18, inhibits mouse 3T6 cell proliferation but not polyomavirus replication

We have purified a cell regulatory sialoglycopeptide, CeReS-18, from intact bovine cerebral cortex cells. This is an 18-kDa molecule that reversibly inhibits cellular DNA synthesis and the proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on mouse 3T6 host cell proliferation and polyomavirus replication was investigated. The results showed that CeReS-18 was able to inhibit 3T6 cell cycling in a concentration-dependent, calcium-sensitive, and reversible manner. Despite the inhibition of cell proliferation, CeReS-18 did not influence polyomavirus infection of 3T6 cells. Indirect immunofluorescent assays revealed that CeReS-18-treated, and cell cycle-arrested, 3T6 cells remained permissive to polyomavirus replication. Electron microscopy and immunogold labeling showed that new viral particles were assembled inside the nuclei of infected cells in the presence of CeReS-18 and during cell cycle arrest. The cellular requirements for the replication of polyomavirus DNA and the synthesis of viral proteins, as well as for the assembly of viral particles, therefore, remained available in CeReS-18-inhibited 3T6 cells. In addition, although polyomavirus infection can be mitogenic, infection of CeReS-18-treated 3T6 cells did not reverse the cell cycle arrest mediated by this cell cycle inhibitor.     

Commitment point during G0-->G1 that controls entry into the cell cycle

Initiation of T-lymphocyte-mediated immune responses involves two cellular processes: entry into the cell cycle (G(0)-->G(1)) for clonal proliferation and coordinated changes in surface and secreted molecules that mediate effector functions. However, a point during G(0)-->G(1) beyond which T cells are committed to enter the cell cycle has not been defined. We define here a G(0)-->G(1) commitment point that occurs 3 to 5 h after CD3 and CD28 stimulation of human CD4 or CD8 T cells. Transition through this point requires cdk6/4-cyclin D, since inhibition with TAT-p16(INK4A) during the first 3 to 5 h prevents cell cycle entry and maintains both naive and memory T cells in G(0). Transition through the G(0)-->G(1) commitment point is also necessary for T cells to increase in size, i.e., to enter the cellular growth cycle. However, transition through this point is not required for the induction of effector functions. These can be initiated while cells are maintained in G(0) with TAT-p16(INK4A). We have termed this quiescent, activated state G(0(A)). Our data provide proof of the principle that entry of T cells into the cell cycle and cellular growth cycles are coupled at the G(0)-->G(1) commitment point but that these processes can be uncoupled from the early expression of molecules of effector functions.     

The effect of factors released from the tumor-transformed cells on DNA synthesis, mitosis, and cellular enlargement in 3T3 fibroblasts

Quiescent serum-starved 3T3 cells can be stimulated to initiate DNA synthesis after addition of conditioned media from spontaneously tumor-transformed 3T3 cells (3T6-cells) or from SV-40-transformed 3T3 cells (SV-3T3 cells). The conditioned media were found to stimulate both the chromosome cycle (i.e., DNA synthesis and cell division) and the growth cycle (i.e., cellular enlargement). Furthermore, addition of conditioned media to quiescent 3T3 cells increased the activity of HMG CoA reductase--an enzyme previously proposed to exercise some control on cell proliferation in 3T3 cells (Larsson and Zetterberg: J. Cell. Physiol. 129:99-102, 1986. The increased activity of HMG CoA reductase after treatment with tumor cell conditioned media was correlated to the stimulatory effects on DNA synthesis. By treating 3T3 cells stimulated to resume proliferation by addition of conditioned media with mevinolin (a competitive inhibitor of HMG CoA reductase) the activity of HMG CoA reductase as well as the DNA synthesis and cell division were efficiently inhibited. In contrast, HMG CoA activity was not coupled to the cellular enlargement. Therefore, it is proposed that one set of factors present in tumor cell conditioned media preferentially stimulates the chromosome cycle by increasing the HMG-CoA reductase activity, whereas another set of factors is responsible for growth in cell size. Both types of factors are required for balanced growth.     

Tbl3 regulates cell cycle length during zebrafish development

The regulation of cell cycle rate is essential for the correct timing of proliferation and differentiation during development. Changes to cell cycle rate can have profound effects on the size, shape and cell types of a developing organ. We previously identified a zebrafish mutant ceylon (cey) that has a severe reduction in T cells and hematopoietic stem/progenitor cells (HSPCs). Here we find that the cey phenotype is due to absence of the gene transducin (beta)-like 3 (tbl3). The tbl3 homolog in yeast regulates the cell cycle by maintaining rRNA levels and preventing p53-induced cell death. Zebrafish tbl3 is maternally expressed, but later in development its expression is restricted to specific tissues. Tissues expressing tbl3 are severely reduced in cey mutants, including HSPCs, the retina, exocrine pancreas, intestine, and jaw cartilage. Specification of these tissues is normal, suggesting the reduced size is due to a reduced number of differentiated cells. Tbl3 MO injection into either wild-type or p53-/- mutant embryos phenocopies cey, indicating that loss of tbl3 causes specific defects in cey. Progression of both hematopoietic and retinal development is delayed beginning at 3 day post fertilization due to a slowing of the cell cycle. In contrast to yeast, reduction of Tbl3 causes a slowing of the cell cycle without a corresponding increase in p53 induced cell death. These data suggest that tbl3 plays a tissue-specific role regulating cell cycle rate during development.     

Effects of growth phase,diel cycle and macronutrient stress on the quantification of Heterosigma akashiwo using qPCR and SHA

The development of molecular probe technologies over the last several decades has enabled more rapid and specific identification and enumeration of phytoplankton species compared to traditional technologies, such as light microscopy. Direct comparisons of these methods with respect to physiological status, however, are sparse. Here we directly compare quantitative real-time PCR (qPCR) and sandwich hybridization assay (SHA) for enumerating the raphidophyte Heterosigma akashiwo at several points during its growth phase, over a diel cycle and with macronutrient stress in laboratory cultures. To ensure consistency between comparisons, a single cellular homogenate was generated from each culture and split for analysis by qPCR and SHA. Since the homogenate was generated from the same number of cells during each experiment, results reflect changes in nucleic acid content (rRNA and DNA) at each time point or in response to environmental conditions relative to a reference sample. Results show a greater level of precision in SHA results which contributed to significant (2–3 fold) differences in rRNA content per cell in several of these analyses. There was significantly greater rRNA content during lag and exponential phases compared to stationary phase cultures, and a significant decrease in rRNA content during the light cycle compared to cells harvested in the dark. In contrast, there were no significant differences in DNA content per cell as determined by qPCR over a diel cycle or during different growth phases. There was also no decrease in either rRNA or DNA content for cultures under low P conditions compared to nutrient replete conditions. However, both rRNA and DNA content were significantly lower under N stress when compared to nutrient replete conditions. Results of this study suggest that growth stage, nutrient stress and cell cycle may impact molecular analyses, and that physiological status should be taken into account when using these methods for HAB monitoring.     

RNA dependence in the cell cycle of V79 cells

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.     

Quantification of Tetradesmus obliquus (Chlorophyceae) cell size and lipid content heterogeneity at single‐cell level

Much of our current knowledge of microbial growth is obtained from studies at a population level. Driven by the realization that processes that operate within a population might influence a population's behavior, we sought to better understand Tetradesmus obliquus (formerly Scenedesmus obliquus ) physiology at the cellular level. In this work, an accurate pretreatment method to quantitatively obtain single cells of T. obliquus , a coenobia‐forming alga, is described. These single cells were examined by flow cytometry for triacylglycerol (TAG ), chlorophyll, and protein content, and their cell sizes were recorded by coulter counter. We quantified heterogeneity of size and TAG content at single‐cell level for a population of T. obliquus during a controlled standard batch cultivation. Unexpectedly, variability of TAG content per cell within the population increased throughout the batch run, up to 400 times in the final stage of the batch run, with values ranging from 0.25 to 99 pg · cell^?1. Two subpopulations, classified as having low or high TAG content per cell, were identified. Cell size also increased during batch growth with average values from 36 to 70 μm³ · cell^?1; yet cell size variability increased only up to 16 times. Cell size and cellular TAG content were not correlated at the single‐cell level. Our data show clearly that TAG production is affected by cell‐to‐cell variation, which suggests that its control and better understanding of the underlying processes may improve the productivity of T. obliquus for industrial processes such as biodiesel production.     

Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition

Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly. We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z. Strezoska, D. G. Pestov, and L. F. Lau, Mol. Cell. Biol. 20:5516-5528, 2000). Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts. In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase. Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased. The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point. Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing. These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle. We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner.     

The relative amounts of the cytoplasmic RNA species in normal, transformed and senescent cultured cell lines.

We have examined the relative quantities of 18S and 28S rRNA, 4S RNA and poly (A) + mRNA in the following cultured cells: the mouse fibroblast lines 3T3 and 3T6 in the resting (contact inhibited) and growing (sparse) states, 3T3 clones transformed with SV40 (SV3T3) and with both SV40 and polyoma SV-Py 3T3), hamster lung fibrobalsts (v79), human cervical carcinoma cells (HeLa), and human diploid fibroblasts at early and late passage. The relative quantities of the RNA species were determined by labeling the cells to equilibrium with 32PO4 and measuring the amount of label in each RNA species. The ratio of mRNA to rRNA varied form 1.1% to 2.7% in the different cell lines, the more rapidly growing cell lines usually giving a higher ratio. In cells experiencing growth limitation either by contact inhibition or due to senescence, the ratio of mRNA to rRNA was about 30% lower than in the corresponding cells in the growing state. In most cell lines the ratio of 4S RNA to 18S rRNA was between 0.8 and 1.2, but in seescent fibroblasts, this ratio increased to greater than 1.7. Senescent fibroblasts also contained much more total RNA per unit of DNA than the same cells at early passage or than 3T6 or 3T3 cells.     

Changes in cell cycle and RNA content in cultured cancer cells treated with cis-diamminedichloroplatinum (II)

Recent reports have shown that CDDP interacts with RNA and protein as well as DNA. We studied the alteration of cell cycle, cellular RNA content and the effect of nucleic acid metabolism on cultured cancer cells after treatment with CDDP by flow cytometry and 3H incorporation assay. The alteration of cell cycle was found to be accumulation of cells in after delay S phase in cytostatic concentrations, CDDP inhibited 3H-TdR uptake markedly at this time and 3H-UR uptake earlier. Increase in RNA content accompanied accumulation of cells in G2M phase. This increase was not a specific phenomenon caused by CDDP, because increase in RNA content was also induced by other inhibitors of DNA synthesis. It is more likely that the direct alteration of cell cycle and cellular RNA content due to action of DNA-combined CDDP rather than that of RNA-combined CDDP.     

Changes in RNA in relation to growth of the fibroblast. I. Amounts of mRNA,rRNA, and tRNA in resting and growing cells

The established cell lines 3T6 and 3T3 contain more of both rRNA and mRNA when they are growing than when they are resting, but mRNA is increased more than rRNA. During conversion of 3T6 cultures from resting to growing state, mRNA, rRNA, and tRNA accumulate long before DNA synthesis begins. The increases in rRNA and tRNA are coordinate, but mRNA accumulates earlier and to a greater extent than the others. The rate of protein synthesis in cultures in transition from resting to growing state increases faster than their content of rRNA and appears proportional to the amount of mRNA rather than to the number of ribosomes. The doubling of mRNA content that takes place before any cells begin to synthesize DNA should be considered in relation to the absence of change in the overall rate of synthesis of HnRNA during the same period.     

Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.     

Quantitative analysis of cytoskeletal proteins throughout the cell cycle of the MRC-5 fibroblastic cell line

Fluorescent dyes were used to stain actin, vimentin, tubulin and DNA in the same MRC-5 fibroblastic cells. Cytofluorometry and image analysis were then used to quantitatively evaluate the F actin, vimentin and tubulin content throughout the cell cycle. The results showed that different cells can have the same DNA content while their cytoskeletal protein content is variable. The data also showed that cytoskeletal protein content variations exist throughout the cell cycle of the fibroblastic cell line. The F actin content increased during the cell cycle from G1 to G2 phases and decreased in M phase. The amount of tubulin in the G2 was about twice as much as that in the G1 phase, before decreasing in the M phase; there was a threshold of tubulin content for G2 cells entering S phase.     

Multiparametric cell cycle analysis by automated microscopy

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.