Effect of mutations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> tumor suppressor <Emphasis Type="Italic">Merlin</Emphasis> on proliferation and differentiation of wing cells

Experiments on transplantation of wing imaginal discs homozygous for a mutation in the tumor suppressor gene Merlin have demonstrated that this mutation does not induce malignant tumors. Marking of the wing disc compartment borders by specific antibodies showed the absence of essential compartment border defects in case of the Merlin mutation. Drosophila melanogaster cells mutant for Merlin have shorter cell cycle than normal cells. Proliferation of imaginal discs lasts longer in case of the mutation. It is known that beginning from some moment of development, wing veins serve as clonal restriction lines that cannot be crossed by growing mosaic clones. We showed that the Merlin mutation leads to depression of vein clonal restriction property. This means that this gene is involved not only in the control of cell proliferation, but also in the control of cell mobility and adhesion.     

Drosophila twin spot clones reveal cell division dynamics in regenerating imaginal discs

Cell proliferation is required for tissue regeneration, yet the dynamics of proliferation during regeneration are not well understood. Here we investigated the proliferation of eye and leg regeneration in fragments of Drosophila imaginal discs. Using twin spot clones, we followed the proliferation and fates of sister cells arising from the same mother cell in the regeneration blastema. We show that the mother cell gives rise to two sisters that participate equally in regeneration. However, when cells switch disc identity and transdetermine to another fate, they fail to turn off the cell cycle and continue dividing long after regeneration is complete. We further demonstrate that the regeneration blastema moves as a sweep of proliferation, in which cells are displaced. Our results suggest that regenerating cells stop dividing once the missing parts are formed, but if they undergo a switch in cell fate, the proliferation clock is reset.     

An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes

Developing cardiac myocytes divide a limited number of times before they stop and terminally differentiate, but the mechanism that stops their division is unknown. To help study the stopping mechanism, we defined conditions under which embryonic rat cardiac myocytes cultured in serum-free medium proliferate and exit the cell cycle on a schedule that closely resembles that seen in vivo. The culture medium contains FGF-1 and FGF-2, which stimulate cell proliferation, and thyroid hormone, which seems to be necessary for stable cell-cycle exit. Time-lapse video recording shows that the cells within a clone tend to divide a similar number of times before they stop, whereas cells in different clones divide a variable number of times before they stop. Cells cultured at 33 degrees C divide more slowly but stop dividing at around the same time as cells cultured at 37 degrees C, having undergone fewer divisions. Together, these findings suggest that an intrinsic timer helps control when cardiac myocytes withdraw from the cell cycle and that the timer does not operate by simply counting cell divisions. We provide evidence that the cyclin-dependent kinase inhibitors p18 and p27 may be part of the timer and that thyroid hormone may help developing cardiac myocytes stably withdraw from the cell cycle.     

Differential division rates and size control in the wing disc

We have generated wing disc compartments that contain marked fast growing M+ clones surrounded by slow dividing M/+ cells. Under these conditions the interactions between fast and slow dividing cells at the clone borders frequently lead to cell competition. However, our assay suppressing apoptosis indicates that cell competition plays no major role in size control. We argue that cells within a compartment proliferate according to their genotype independently of each other and that their contribution to the final structure will depend solely on their proliferation rate. This model is supported by a computer simulation that predicts values similar to those found experimentally. Our results on the growth of M+ clones within compartments and on the expression of developmental genes like vestigial and wingless suggest the existence of a non-cell autonomous mechanism that functions at the level of the entire cell population. It measures the population size in each moment, determines the corresponding expression levels of developmental genes and establishes the time to arrest growth.     

Duration of the cell cycle phases in mutants for the tumor suppressor Merlin in Drosophila melanogaster

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 pulse-labeled 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.     

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.     

Plant casein kinases phosphorylate and destabilize a cyclin-dependent kinase inhibitor to promote cell division

Cell cycle is one of the most fundamentally conserved biological processes of plants and mammals. Casein kinase1s (CK1s) are critical for cell proliferation in mammalian cells; however, how CK1s coordinate cell division in plants remains unknown. Through genetic and biochemical studies, here we demonstrated that plant CK1, Arabidopsis (Arabidopsis thaliana) EL1-like (AELs), regulate cell cycle/division by modulating the stability and inhibitory effects of Kip-related protein6 (KRP6) through phosphorylation. Cytological analysis showed that AELs deficiency results in suppressed cell-cycle progression mainly due to the decreased DNA replication rate at S phase and increased period of G2 phase. AELs interact with and phosphorylate KRP6 at serines 75 and 109 to stimulate KRP6’s interaction with E3 ligases, thus facilitating the KRP6 degradation through the proteasome. These results demonstrate the crucial roles of CK1s/AELs in regulating cell division through modulating cell-cycle rates and elucidate how CK1s/AELs regulate cell division by destabilizing the stability of cyclin-dependent kinase inhibitor KRP6 through phosphorylation, providing insights into the plant cell-cycle regulation through CK1s-mediated posttranslational modification.

Plant casein kinases coordinate cell cycle by regulating the stability of a cyclin-dependent kinase inhibitor through promoting interaction with E3 ubiquitin ligases and proteasomal degradation by phosphorylation.     

The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila

Brain development in Drosophila is characterized by two neurogenic periods, one during embryogenesis and a second during larval life. Although much is known about embryonic neurogenesis, little is known about the genetic control of postembryonic brain development. Here we use mosaic analysis with a repressible cell marker (MARCM) to study the role of the brain tumor (brat) gene in neural proliferation control and tumour suppression in postembryonic brain development of Drosophila. Our findings indicate that overproliferation in brat mutants is due to loss of proliferation control in the larval central brain and not in the optic lobe. Clonal analysis indicates that the brat mutation affects cell proliferation in a cell-autonomous manner and cell cycle marker expression shows that cells of brat mutant clones show uncontrolled proliferation, which persists into adulthood. Analysis of the expression of molecular markers, which characterize cell types in wild-type neural lineages, indicates that brat mutant clones comprise an excessive number of cells, which have molecular features of undifferentiated progenitor cells that lack nuclear Prospero (Pros). pros mutant clones phenocopy brat mutant clones in the larval central brain, and targeted expression of wild-type pros in brat mutant clones promotes cell cycle exit and differentiation of brat mutant cells, thereby abrogating brain tumour formation. Taken together, our results provide evidence that the tumour suppressor brat negatively regulates cell proliferation during larval central brain development of Drosophila, and suggest that Prospero acts as a key downstream effector of brat in cell fate specification and proliferation control.     

A lymphocyte blastogenesis inhibitory factor (LBIF) reversibly arrests a human melanoma cell line, A375, at G1 and G2 phases of cell cycle

A lymphocyte blastogenesis inhibitory factor, LBIF, has been found in the culture supernatant of a human macrophage-like cell line, U937. The factor has been purified by fast protein liquid chromatography. Partial amino acid sequencing analysis showed that LBIF was a novel immunoregulatory factor. Recent study has demonstrated that LBIF possesses a remarkable tumor growth inhibitory activity. In this study, the cell growth inhibitory activity of LBIF was characterized on the proliferation of a human melanoma cell line A375 in vitro. LBIF strongly inhibits the proliferation of A375 cells. The inhibitory activity was cytostatic and reversible by Day 5 although the lethal effect became apparent at Day 7. Cell cycle analysis by flow cytometry showed that LBIF arrested A375 cells at both G1 and G2/M phases. Mitotic index analysis indicated that A375 cells were arrested in G1 and G2 phases. LBIF function was not attributed to the elevation of intracytoplasmic cyclic-AMP levels. Thus, these results suggest that LBIF plays an important role in controlling cell cycle and there is a similarity between the mechanisms of G1 and G2 arrests in eukaryotic cell proliferation. LBIF-induced reversible cell-cycle arrest of A375 cells can be a useful system to analyze the signal transduction for cell proliferation and cell-cycle arrest.     

Mouse epidermal stem cells proceed through the cell cycle

The epidermis is a continuously renewing tissue maintained by undifferentiated stem cells. For decades it has been assumed that epidermal stem cells (ESCs) were held in the G0 phase of the cell cycle and that they only entered the cell cycle when needed. Previously, we showed that ESCs retained nuclear label for long periods, indicating that these cells did not proceed through the cell cycle at the same rate as the other proliferative basal cells. However, their exact cell-cycle profile has not been determined because a pure population of ESCs has not been available. In this study, we sorted stem and transient amplifying (TA) cells from murine neonatal back skin, and adult ear, footpad, and back skin, using our recently developed method. We found that neonatal back skin had two times the number of ESCs as the adult tissues. Despite the age and anatomical difference, these ESC populations exhibited similar cell cycle profiles with approximately 96% in G0/G1 and 4% in S-G2/M. The cell cycle profiles of the TA cells from neonatal back skin and adult footpad also showed a profile similar to each other (85% in G1 and 15% in S-G2/M). Examination of genes on a cell cycle chip showed that proliferation associated genes and only p57 were upregulated in the TA cell and ESC population, respectively. We found BrdU positive and cyclin B1 positive cells in all groups, confirming that both ESCs and TA cells were cycling. These data demonstrate that there are more TA cells dividing than ESCs, that the cell cycle profile of adult TA cells is related to the proliferative state of the tissue in which they reside, and that ESC proceed through the cell cycle.     

MCPIP1 overexpression in human neuroblastoma cell lines causes cell-cycle arrest by G1/S checkpoint block

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.     

Parameters of cell competition in the compartments of the wing disc of Drosophila.

When compartments in the wing disc of Drosophila are mosaic for two populations of cells, one of which is dividing more slowly, then the slower-growing population tends to be eliminated. The phenomenon leading to the disappearance of nonlethal, slowly dividing Minute cells was termed cell competition by Morata and Ripoll (1975) [Morata, G., and Ripoll, P. (1975). Develop. Biol.427, 211–221]. In this paper the different parameters of cell competition are explored. Starvation of the larvae rescued the Minute clones and permitted the following observations: The Minute clones grow to some extent before being out-competed. Prior to their disappearance, they become fragmented into small patches. Cell competition is greater in the centre of compartments than in the boundary regions. Possible causes of cell competition are discussed, as well as the hypothesis that the phenomenon may be related to the control of growth.     

Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells

Skeletal muscle retains a resident stem cell population called satellite cells, which are mitotically quiescent in mature muscle, but can be activated to produce myoblast progeny for muscle homeostasis, hypertrophy and repair. We have previously shown that satellite cell activation is partially controlled by the bioactive phospholipid, sphingosine-1-phosphate, and that S1P biosynthesis is required for muscle regeneration. Here we investigate the role of sphingosine-1-phosphate receptor 3 (S1PR3) in regulating murine satellite cell function. S1PR3 levels were high in quiescent myogenic cells before falling during entry into cell cycle. Retrovirally-mediated constitutive expression of S1PR3 led to suppressed cell cycle progression in satellite cells, but did not overtly affect the myogenic program. Conversely, satellite cells isolated from S1PR3-null mice exhibited enhanced proliferation ex-vivo. In vivo, acute cardiotoxin-induced muscle regeneration was enhanced in S1PR3-null mice, with bigger muscle fibres compared to control mice. Importantly, genetically deleting S1PR3 in the mdx mouse model of Duchenne muscular dystrophy produced a less severe muscle dystrophic phenotype, than when signalling though S1PR3 was operational. In conclusion, signalling though S1PR3 suppresses cell cycle progression to regulate function in muscle satellite cells.     

Proliferation controls in a linear growth system: theoretical studies of cell division in the cartilage growth plate

The columnar arrangement of dividing cells in the epiphyseal cartilage plates of growing bones provides a model of a linear proliferation system. One factor which determines the rate of cell production, and hence the rate of growth, is the size of the proliferating population. In this one dimensional system this size is equal to the length of the proliferation zone. Two possible mechanisms for a differentiation control that sets a limit to the length of this zone have been tested in computer simulations. While a diffusion gradient control is consistent with cell kinetic measurements a division limit based on an inheritable growth substance is shown to require further development before the model fits experimental data.Cell division in the columns produces linear clones of cells. If the final length of a bone is set by a limit on the number of divisions that the cartilage stem cells can make, then the number of cells per clone is crucial in determining overall bone growth. The parameters that affect linear clone size have been investigated in computer simulations. Clone size depends largely on the relative division rate of stem cells to proliferation zone cells — but the data on stem cell division rates are generally unreliable.The analysis could be applied to other linear proliferating systems.     

Arrest of Chinese hamster cells in G 2 following treatment with the anti-tumor drug bleomycin

Upon addition of bleomycin (BLM) to suspension cultures of Chinese hamster cells (line CHO), cells closer to prophase than 56 minutes continue dividing at the normal rate, whereas cells at earlier positions in the cell cycle either fail to reach mitosis altogether (at 200 μg/ml) or enter mitosis and divide at a reduced rate at lower drug concentrations. At 100 μg/ml of BLM (the rate of cell division slowed to a doubling time of 167 hours), initiation and termination of DNA synthesis occur at normal rates, resulting in an accumulation of cells with a G₂ DNA content in the first 130 minutes of G₂. Bleomycin effects are not readily reversible. The rates of incorporation of leucine, uridine, or thymidine into cells treated for six hours with 100 μg/ml of BLM were 90, 85, and 80%, respectively, of the values obtained in control cultures, suggesting that the effects of BLM on cell-cycle traverse cannot be correlated with gross inhibition of macromolecular synthesis.     

Abnormalities in cell proliferation and apico-basal cell polarity are separable in Drosophila lgl mutant clones in the developing eye

In homozygous mutants of Drosophila lethal-2-giant larvae (lgl), tissues lose apico-basal cell polarity and exhibit ectopic proliferation. Here, we use clonal analysis in the developing eye to investigate the effect of lgl null mutations in the context of surrounding wild-type tissue. lgl⁻ clones in the larval eye disc exhibit ectopic expression of the G1-S regulator, Cyclin E, and ectopic proliferation, but do not lose apico-basal cell polarity. Decreasing the perdurance of Lgl protein in larval eye disc clones, by forcing extra proliferation of lgl⁻ tissue (using a Minute background), leads to a loss in cell polarity and to more extreme ectopic cell proliferation. Later in development at the pupal stage, lgl mutant photoreceptor cells show aberrant apico-basal cell polarity, but this is not associated with ectopic proliferation, presumably because cells are differentiated. Thus in a clonal context, the ectopic proliferation and cell polarity defects of lgl⁻ mutants are separable. Furthermore, lgl⁻ mosaic eye discs have alterations in the normal patterns of apoptosis: in larval discs some lgl⁻ and wild-type cells at the clonal boundary undergo apoptosis and are excluded from the epithelia, but apoptosis is decreased elsewhere in the disc, and in pupal retinas lgl⁻ tissue shows less apoptosis.     

Co-cultivation of whole blood from male and female muntjacs and the cell proliferation kinetics in vitro

A mixed blood culture (MBC) of heparinized whole blood from male and female Indian muntjac has been done using the BrdU-Hoechst-sunlight-Giemsa method to study the cell-cycle kinetics in vitro. Blood lymphocytes of both male and female muntjacs show a much shorter cell cycle time, roughly, 10-12 h for the initial but only 8 h for the subsequent cycles. There is a significant difference in the rate of cell proliferation between male and female cells. The male blood cells constitute a majority of the 'slow'-dividing cells which reach a peak at the first cycle of mitosis at 40 h, whereas a similar peak of first cycle mitosis is reached by female cells at 32 h, indicating the occurrence of a high frequency of 'fast' dividing female cells as compared to those of males. This novel sex-based differential cell proliferation kinetics is observed both in mixed and separate cultures. This type of MBC method which is free of interculture variations can be reliably used for comparative studies where two genomes can be distinguished.