Growth-mediated, density-dependent inhibition of endocytosis in cultured arterial smooth muscle cells

The effects of cell density and growth upon fluid phase endocytosis were investigated in quiescent and growing cultures of monkey arterial smooth muscle cells. Cells were maintained in a quiescent state of growth in 5% plasma-derived serum. Subsequent exposure of subconfluent cultures to the specific mitogens, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), or to whole blood serum, resulted in up to 4-fold increases in the rate of fluid endocytosis/cell. The changes began several hours after entry into G1 phase of the cell cycle and continued through S. The fraction of cells entering the growth cycle was variable (PDGF=FGF>EGF) and a close correlation existed between the rate of endocytosis and the fraction of [³H]thymidine-labelled cells (r = 0.929, p<0.01). At a range of cell densities, the rate of fluid endocytosis/cell was similar in sparse, confluent and post-confluent cultures of quiescent cells; in contrast, in growing cells there was density-dependent inhibition of endocytosis. Furthermore, when quiescent cells were in contact with each other and were then exposed to mitogens, the growth response was diminished and there was only a 25–50% increase in the rate of endocytosis, even in the presence of high concentrations of growth factors.These studies indicate that the influence of cell density upon fluid endocytosis in arterial smooth muscle cells is indirect in that it represents a secondary effect of decreased mitogenic response to specific growth factors.     

Density-dependent inhibition of 2-deoxy-d-glucose uptake into glioma and neuroblastoma cells in culture

The transport of 2-deoxy-d-glucose (2-DG) into cultured human glioma (138 MG) and mouse neuroblastoma (C1300) cells has been studied in relation to the growth curves of the cells. An initial increase in the uptake of 2-DG into exponentially growing 138 MG cells, could be attributed to the number of days the cells were kept in culture rather than to their increased density. As the 138 MG cells reached confluency after 3 days the 2-DG uptake became density-dependent inhibited. In still denser cultures the cell growth was inhibited. This was accompanied by morphological ‘normalization’ of the cells and increased uptake of 2-DG. Uptake of 2-DG into C1300 cells was density-dependent inhibited throughout the cell growth cycle. As the cell density increased from 15 × 10³ to 130× 10³ cells/cm² the rate of uptake/cell decreased to one-fourth. At the latter cell density the cells entered stationary phase, without any major changes in morphology. The results suggest that spontaneously occurring tumour cells, such as glioma and neuroblastoma cells, can regulate the sugar transport in relation to cell density. This could be due to newly-acquired differentiated properties of the cells or to true contact-inhibitory phenomena.     

Cell-cell contact and growth regulation of pinocytosis in 3T3 cells

In sub-confluent cultures of Balb/c-3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet-derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth-inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density-inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40-viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density-dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth-inhibition rather than to any direct physical effects of cell–cell contact.     

p27 modulates cell cycle progression and chemosensitivity in human malignant glioma.

The cell cycle regulatory protein p27, an inhibitor of cyclin-dependent kinases (CDK), has been attributed a role in (i) prognosis in breast and colon cancer, (ii) induction of apoptosis in cancer cells, and (iii) resistance to cancer chemotherapy. Here we report that p27 is widely expressed in human malignant gliomas in vivo and in glioma cell lines in vitro. Serum deprivation or confluency promotes p27 protein accumulation in vitro. Neither baseline p27 levels nor p27 levels induced by confluency or serum deprivation correlate with p53 status or drug sensitivity of human glioma cell lines. Expression of antisense p27 mRNA increased the doubling times in T98G glioma cells, whereas sense p27 mRNA had no such effect. There was a density-dependent and drug-specific modulation of chemosensitivity by sense or antisense mRNA expression in T98G cells. Taken together, these data define a strong p27 response to altered growth conditions and suggest a role for p27 in modulating response to chemotherapy in human malignant glioma cells.     

Characteristics and Living Patterns of Marine Myxobacterial Isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.     

Serum stimulation of stationary human glia and glioma cells in culture

Stationary cell cultures can be stimulated to re-enter the cell cycle by giving medium containing fresh serum. The mechanism for this initiation was studied in normal human glia and neoplastic glioma cells in order to demonstrate any differences between normal and neoplastic cells regarding the kinetics of their march through G 1 into the S phase of the cell cycle. Glia cells were found to demand at least 12 h of serum stimulation to enter the cell cycle and were committed to DNA synthesis about 4 h prior to the actual onset of this synthesis. On the other hand, as little as l h of serum stimulation induced DNA synthesis in a fraction of glioma cells and these malignant cells were committed during the largest part of the prereplicative phase. This difference in commitment kinetics could not be explained by differences in cell cycle parameters or medium deficiencies and is considered to reflect a part of the fundamental difference between normal and neoplastic cells as regards their growth control.     

Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture

The effects of vitamin K on the morphology and the growth of mouse neuroblastoma (P₂), mouse melanoma (B-16) and rat glioma (C-6) cells in culture were studied. Vitamin K₃ inhibited the growth (due to cell death and partial or complete inhibition of cell division) of all three cell types without causing any morphological differentiation. Vitamin K₃ was more effective than vitamin K₁. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma or glioma cells. Glioma cells did not grow in hormone-supplemented serum-free medium; however, both neuroblastoma and melanoma cells grew to a level 70–80% of that found in serum-supplemented medium. Neuroblastoma cells and melanoma cells cultured in serum-free medium exhibited a 2–3 fold higher sensitivity to vitamin K₃ than those cultured in serum-supplemented medium. This suggests that serum factors attenuate the growth inhibitory effect of vitamin K₃ on tumor cells in culture, probably by reducing the availability of this vitamin to the cells. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma cells even when they were treated in serum-free medium. The fact that micromolar concentrations of vitamin K₃ inhibit the growth of tumor cells in culture suggests that this vitamin may be a potentially useful anticancer agent.     

Changes in p21(Cip1) and p27(Kip1) expression are not required for cell cycle entry and progression to S phase in Swiss 3T3 cells

The cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), play an important role in the regulation of progression through G(1) to S phase in mammalian cells. Here we report that confluent 3T3 cells expressed p21(Cip1) and p27(Kip1) predominantly in the nucleus, and the level of both proteins declined as the cells entered the cell cycle and progressed through G(1) in response to serum growth factors. However, when confluent cells were serum starved prior to treatment, no downregulation of p21(Cip1) or p27(Kip1) expression was observed. Notably, serum starvation did not significantly influence the capacity of the cells to progress to the S phase. It was observed that serum starvation reduced cell density. Further, when cells were plated at a range of different densities, starved of serum to render them quiescent and then subsequently treated with serum, a reduction in p21(Cip1) and p27(Kip1) expression was observed in cells plated at high density but not in those at low density. Again, the extent and timing of progression to S phase was not influenced by cell density. To establish the potential role of cell:cell contact in the observed density-dependent regulation of p21(Cip1) and p27(Kip1) expression, cells were plated onto micorarrays of adhesive islands that prevented individual cells from making any contact with other cells. Under these conditions serum growth factors induced p21(Cip1) and p27(Kip1) downregulation, and hence, there is no requirement for cell:cell contact. Together, these data indicate that there are conditions under which 3T3 cells can progress to the S phase without downregulation of p21(Cip1) and p27(Kip1). The significance of these observations and mechanisms by which density-dependent regulation of p21(Cip1) and p27(Kip1) expression may occur are discussed.     

Cell density downregulates DNA synthesis and proliferation during osteogenesis in vitro

Abstract. The classical models of in vitro cell culture comprise fibroblasts and epithelial cells. Osteogenic cells represent another interesting cell model; however, it is not known whether during osteogenesis cell density regulates cell growth as seen in cultures of fibroblasts and epithelial cells. We selected MC3T3-E1 cells for study because they are an osteogenic cell line that, when subcultured, grow to confluence and form multilayers of cells in conventional cultures by continued proliferation, as do fibroblasts. Once maximum cell density is obtained, proliferation is down regulated resulting in a mixed population of quiescent and dividing cells. We used this model to determine whether downregulation of proliferation as expressed by cell number and DNA synthesis is cell density-dependent. MC3T3-E1 cells were cultured over a period of 34 days to determine their kinetics, viability, ability to synthesize DNA, distribution within phases of the cell cycle and cell number-response relationships. Our results show that (1) viability ranged between 92% and 96% and the cell number 2.5 x 10⁵ per cm² once cultures reached steady state, (2) most cells entered the G₀/G₁ phase of the cell cycle on day 7, (3) there was no correlation between the proportion of cells in S phase and downregulation of DNA synthesis, (4) a direct relationship exists between cell density and downregulation of DNA synthesis on day 8, (5) the minimum time for cells to be cultured before downregulation of DNA synthesis begins is independent of cell number, and (6) downregulation of DNA synthesis is reversible. These results suggest that density-dependent downregulation of DNA synthesis may be a mechanism of growth control for osteogenic cells in vitro that operates more like density-dependent growth control in cultures of fibroblasts rather than epithelial cells.     

Non-sufficient cell cycle control as possible clue for the resistance of human malignant glioma cells to clinically relevant treatment conditions

Summary. Objectives. Human gliomas have a catastrophic prognosis with a median survival in the range of one year even after therapeutic treatment. Relatively high resistance towards apoptotic stimuli is the characteristic feature of malignant gliomas. Since cell cycle control has been shown to be the key mechanism controlling both apoptosis and proliferation, this study focuses on DNA damage analysis and protein expression patterns of essential cell cycle regulators P53 and P21^waf1/cip1 in glioma under clinically relevant therapeutic conditions. Material and methods. U87MG cell line, characterised by wild p53-phenotype relevant for the majority of primary malignant glioblastomas, was used. Glioma cells underwent either irradiation or temozolomide treatment alone, or combined radio/chemo treatment. DNA damage was analysed by the “Comet Assay”. Expression rates of target proteins were analysed using “Western-Blot” technique. Results and conclusions. “Comet Assay” demonstrated extensive DNA damage caused by temozolomide treatment alone and in combination with irradiation, correlating well with the low survival rate observed under these treatment conditions. In contrast, irradiation alone resulted in a relatively low DNA damage, correlating well with a high survival rate and indicating a poor therapeutic efficiency of irradiation alone. Unusually low up-regulation of P53 and P21^waf1/cip1 expression patterns was produced by the hereby tested stressful conditions. A deficit in cell cycle control might be the clue to the high resistance of malignant glioma cells to established therapeutic approaches.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.     

Potent inhibition of cell density-dependent apoptosis and enhancement of survival by dimethyl sulfoxide in human myeloblastic HL-60 cells

Human myeloblastic cell line HL-60 cells undergo apoptosis during in vitro culture in a cell density-dependent manner, and this cell density-dependent apoptosis was observed when the concentration of cultured cells exceeded 8–10 × 10⁵ cells/ml. Dimethyl sulfoxide (DMSO), a differentiation inducer of HL-60 cells, did not amplify, but rather potently inhibited, this apoptosis. In a low density culture condition, DMSO attenuated proliferation of HL-60 cells in spite of its inhibition of apoptosis. In contrast, DMSO did support cell survival under high cell density conditions, and DMSO-treated HL-60 cells reached an extremely high concentration of 2–3 × 10⁶ cells/ml, a condition which could never be possible in a usual culture environment. Thus, DMSO exerted dual effects on cell proliferation, i.e., growth inhibition and apoptosis inhibition, and the sum of these effects resulted in an apparently distinct phenomenon according to the culture conditions including cell density. J. Cell. Physiol. 174:135–143, 1998. © 1998 Wiley-Liss, Inc.     

Establishment and Growth Kinetics of the Mutant Ehrlich-Lettré Ascites Cell Strain Hd33 In Permanent Suspension Culture1,2

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 × 10^-5 of the explanted cells continued to grow in vitro. the resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. the corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. the duration of the G₁ phase was effectively zero. Both PLM data and [³H]/[¹⁴C] thymidine double-labetling measurements revealed an S-phase duration of between 11 and 12 hr. the G₂ phase lasted 3–5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

Characteristics and living patterns of marine myxobacterial isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.     

Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

Abstract.  Objectives : Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca²⁺ influx pathway impacting cellular growth. Materials and Methods : Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. Results : We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl₃, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells' resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G₂+ M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. Conclusions : Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme , the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours.     

Inhibition of sphingosine kinase 1 suppresses proliferation of glioma cells under hypoxia by attenuating activity of extracellular signal-regulated kinase

Objectives: Sphingosine kinase (SphK), which is regulated by hypoxia, catalyses phosphorylation of sphingosine to produce sphingosine‐1‐phosphate, which stimulates invasiveness of gliomas. However, whether SphK is involved in proliferation of glioma cells under hypoxic conditions is not clearly understood. In this study, we have investigated the role of SphK in of proliferation glioma cells under hypoxia. Materials and methods: Effects of small interfering RNA (siRNA) on SphKs, SKI (inhibitor of SphK) and U0126 (inhibitor of ERK) on proliferation of glioma cells under hypoxia were studied using CCK‐8 assay and flow cytometry. Protein expression profiles were evaluated by Western blot analysis. Results:  SKI suppressed proliferation of glioma cells under hypoxia. Similarly, downregulation of SphKs by siRNA inhibited glioma cell proliferation, and the cell cycle was arrested in G₂/M phase when SphK1 was inhibited. In addition, inhibition of SphK1 attenuated phosphorylation of ERK in hypoxic conditions. Furthermore, U0126 markedly inhibited cell population growth and arrested cells in G₂/M as effectively as SKI. However, silencing SphK2 induced cell cycle arrest in the S phase and it showed little effect on hypoxia‐induced activation of ERK. Conclusions: SphK1 and SphK2 are involved in proliferation of glioma cells in hypoxic conditions through distinct signalling pathways. SphK1, but not SphK2, promotes cell population expansion in hypoxic conditions by activating ERK.     

High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27(Kip) Cdk-complexes

The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells ceased to grow exponentially, to reveal the molecular basis for Ras-dependent focus formation. As normal cells entered density-dependent arrest, cyclin D1 decreased while cyclin D2 was induced and replaced D1 in Cdk4 complexes. Concomitantly, p27(Kip1) levels rose and the inhibitor accumulated in both Cdk4 and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27(Kip1) induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal response to contact inhibition, a separate back-up mechanism enforced cell cycle arrest at higher cell density.     

Effect of imposed serum deprivation on growth of the mouse 3T3 cell. Dissociation from changes in potassium ion transport as measured from [36Rb]rubidium ion uptake

Decreased serum concentrations that substantially alter the growth of normal 3T3 cells alter neither the active and non-active components of unidirectional ⁸⁶Rb⁺ influx nor the intracellular K⁺ content when compared with cells in exponential growth. Thus the changes in K⁺ transport (measured with ⁸⁶Rb⁺ as an analogue for K⁺ movements) that occur on density-dependent growth inhibition of the mouse 3T3 cell are not mimicked by serum deprivation of the cells before density inhibition.     

p53 mediates density-dependent growth arrest

While the stress-response-associated importance of the p53 tumor suppressor is well established, recent studies have also linked p53 with several basic parameters in the normal behavior of cells. Here, we present evidence that basal p53 expression in WI38 human embryonic lung fibroblasts restricts growth rate and mediates density-dependent inhibition of growth and the associated G1 phase arrest of the cell cycle by affecting the density-dependent regulation of p16/INK4a. Additionally, we show that prolonged culturing of hTert-immortalized WI38 cells leads to a loss of density-dependent growth inhibition that correlates with p27/KIP deregulation as well as the previously shown INK4a locus silencing, and to an onset of contact-induced, p53-dependent cell death.     

Changes in RNA Synthesis during the Culture Growth Cycle of Tetrahymena

During logarithmic growth of the holotrichous ciliate Tetrahymena pyriformis , as well as in most cell systems, characteristic growth parameters are constant and highly reproducible. If the cells are allowed, however, to perform the complete culture growth cycle, their entry into the stationary phase is accompanied by events too complex to be attributed to "cessation of proliferation". At that stage we observed a sharp increase in uptake and incorporation of tritiated uridine. This seems to be caused by the increase in specific radioactivity of the external precursor supply following consumption of the non-radioactive counterpart. Density gradient centrifugation of pulse-labelled RNA revealed the cessation of ribosomal RNA synthesis at stationary phase to be due to a transition of pulse-labelled RNA from ribosomal to heterogenous low molecular weight RNA profiles. The decrease in ribosomal and the appearance of heterogenous RNA are discussed in terms of degradation and density-dependent selective inhibition of ribosomal RNA.
Evidence of a density-dependent inhibition of culture growth would indicate principles of growth control functioning in a similar way in unicellular organisms as observed in epithelial cells.