Transferrin receptor expression during exponential and plateau phase growth of human tumour cells in culture

Transferrin receptor expression by the human tumour cell lines CCRF-CEM leukaemia and PMC-22B melanoma was studied, measuring the specific binding of fluorescein isothiocyanate (FITC)-labelled transferrin using a fluorescence-activated cell sorter. By measuring the fluorescence of cells stained at subsaturating concentrations of conjugate it was possible to calculate the average numbers of receptors per cell and the binding affinity by Scatchard analysis. These values (1.9 × 10⁵ binding sites/cell, K^A 1.2 × 10⁹ M^?1 for CCRF-CEM during exponential growth and 6.9 × 10⁴ binding sites/cell, K_A 1.4 × 10^?9 M^?1 for PMC-22B) are in close agreement with previously published data obtained using radiolabelled transferrin. The present method, however, allowed the transferrin receptor expression of individual cells within a population to be measured and thus it has been possible to test the hypothesis that transferrin receptor is a marker for cycling cells. Frequency-distribution histograms of transferrin receptor showed a wide range of values for both cell lines during exponential growth. When the extreme ranges were sorted and the cells examined for cellular DNA content it was found that those with the highest transferrin receptor expression were enriched with cells in S, G₂, and M phases of the cell cycle, whereas those with low transferrin receptor expression were mainly in G₁. However, two-parameter-correlated dot plots of transferrin receptor expression versus DNA content showed there was considerable overlap between the ranges of receptor expression for the different cell cycle compartments. Using a stathmokinetic method we have measured the proportion of quiescent cells in fed plateau phase cultures. Transferrin receptor expression was downgraded under these growth conditions but, contrary to expectation, the decline affected the population uniformly, without the emergence of a distinct, transferrin receptor-negative subpopulation corresponding to the increasing proportion of quiescent cells. Thus, although transferrin receptor expression bears some relation to cell cycle phase and reflects the proliferative activity of populations of cells, it is incapable of identifying individual cells which are out of cycle.     

Relationship between cytoplasmic pH and proliferation during exponential growth and cellular quiescence

Flow cytometry was used to measure intracellular pH (pHi) on an individual cell basis during exponential and plateau phases of growth. In all three cell lines examined a range of pHi values was associated with exponential growth. When cells from the extremes of the pHi distribution were sorted using a fluorescence-activated cell sorter and then restained for cellular DNA content, it was found that the higher pHi values were associated with enrichment of the S, G2, and M phases of the cell cycle, with a corresponding increase in the percentage of G1 cells at the lower pH1 range, suggesting cell-cycle dependence of pHi. It has been shown previously (I. W. Taylor and P. Hodson, 1984, J. Cell Physiol. 121, 517) that PMC-22 human melanoma cells are capable of entering a distinct pH-dependent quiescent state in response to the acidification of the growth medium which occurs naturally during growth to plateau phase. Simultaneous measurement of pHi and external pH showed that under these conditions pHi was maintained at control values down to an external pH of approximately 6.5, below which cytoplasmic acidification took place. This fall in pHi coincided with the onset of the transition to quiescence. Individual quiescent cells (defined by failure to incorporate bromodeoxyuridine during a 24-h exposure) could not be identified as such on the basis of a low pHi, suggesting that the probability of cell cycling is reduced by lowering pHi. Those cells which remained in cycle showed a markedly reduced rate of DNA synthesis, but a cell-cycle phase distribution similar to that in exponential growth, indicating that prolongation of all cell-cycle phases is an additional factor influencing overall population growth. The external pH at which both of these effects on cell proliferation kinetics took place in vitro is similar to that which occurs regionally within solid tumors, suggesting that pH effects could play a significant role in determining tumor cell growth in vivo.     

Murine Mammary Tumour Cells In Vitro. I. the Development of A Quiescent State

Abstract Three mouse mammary tumour lines (66, 67, and 68H) derived from a single mouse mammary tumour were investigated for their growth kinetics and development of quiescent cells in unfed monolayer cultures. All three lines develop pure quiescent populations when grown in unfed plateau cultures. A dramatic cell-cycle redistribution accompanied the proliferating (P) to quiescent (Q) transition, with the percentage of cells having a G₁ DNA content increasing from 50% in the P state to <97% in the Q state. As the cultures progressed from exponential to plateau growth, a decrease of 50% in cellular RNA was observed in all three lines. This property enables the clear identification of P v. Q cells by flow cytometry using the two-step acridine orange assay. Autoradiographic data verified that these plateau cells were quiescent since >2.5% of the cells incorporated [³H]TdR when labelled for approximately two doubling times. Further comparison of the P and Q cells showed that: (a) the Coulter volume of Q cells was approximately half that of P cells in all three lines; (b) viability, as measured by dye exclusion was >95% in all cultures regardless of their proliferative state; and (c) colony-forming ability decreased as the cells entered the quiescent state. In each of these cell lines the development of Q-cell populations was marked by similar changes in all measured parameters. These quiescent tumour cells provide a relatively simple model to evaluate what, if any, important differences exist between the response of P v. Q cells to various therapeutic agents.     

Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

THE PROLIFERATION KINETICS OF NHIK 1922 CELLS IN VITRO AND IN SOLID TUMOURS IN ATHYMIC MICE

The proliferation kinetics of cells of the line NHIK 1922 grown in vitro and as solid tumours in the athymic mutant nude mouse has been studied. In vitro, growth curves were determined for exponentially growing populations and for populations synchronized by mitotic selection. The phase durations for these populations were determined by flow cytofluorometric measurements of DNA-histograms and pulsed incorporation of [³H]TdR respectively. The generation time and the phase durations for synchronized populations were found to be about equal to those for exponentially growing populations. The duration of the phases G₁, S and G₂+ M was found to be 8·5–9·5, 11·0–12·0 and 6·0–6·5 hr respectively, i.e. the generation time was 26·5–27·0 hr. The proliferation kinetics in vivo were studied by flow cytofluorometry and by the technique of percentage labelled mitoses. The median duration of S-phase and (G₂+ M)-phase in vivo was found to be approximately the same as that observed in vitro, while the median duration of G₁-phase was found to be approximately 5 hr longer in vivo than under the present in vitro growth conditions. The growth fraction in vivo was estimated to be approximately 50%. The non-proliferative compartment of the tumour cells was found to consist mainly of cells with the DNA-content of cells in G₁-phase. It is concluded that the reduced rate of proliferation of NHIK 1922 cells in vivo is correlated with alterations in the duration of G₁-phase and, hence, the proportion of cells in G₁-phase.     

Quiescence in 9L cells and correlation with radiosensitivity and PLD repair

The onset of quiescence, changes in X-ray sensitivity, and changes in capacity for potentially lethal damage (PLD) repair of unfed plateau-phase 9L44 cell cultures have been systematically investigated. The quiescent plateau phase in 9L cells was the result of nutrient deprivation and was not a cell contact effect. Eighty-five to 90% of the plateau-phase cells had a G1 DNA content and a growth fraction less than or equal to 0.15. The cell kinetic shifts in the population were temporally correlated with a developing radioresistance, which was characterized by a larger shoulder in the survival curve of the quiescent cells (Dq = 5.71 Gy) versus exponentially growing cells (Dq = 4.48 Gy). When the quiescent plateau-phase cells were refed, an increase in radiosensitivity resulted which approached that of exponentially growing 9L cells. Delayed plating experiments after irradiation of exponentially growing cells, quiescent plateau-phase cells, and synchronized early to mid-G1-phase cells indicated that while significant PLD repair was evident in all three populations, the quiescent 9L cells had a higher PLD repair capacity. Although data for immediate plating indicated that 9L cells may enter quiescence in the relatively radioresistant mid-G1 phase, the enhanced PLD repair capacity of quiescent cells cannot be explained by redistribution into G1 phase. When the unfed quiescent plateau-phase 9L cells were stimulated to reenter the cell cycle by replating into fresh medium, the first G1 was extended by 6 h compared with the G1 of exponentially growing or refed plateau-phase 9L cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Murine Mammary Tumour Cells In Vitro. Ii. Recruitment of Quiescent Cells

Abstract The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. the length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state. however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach 50% were incapable of either increasing their RNA levels to that of proliferating G₁ cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Slnce clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

Regulation of human ornithine decarboxylase expression following prolonged quiescence: Role for the c-Myc/Max protein complex

WI-38 cells can remain quiescent for long periods of time and still be induced to reenter the cell cycle by the addition of fresh serum. However, the longer these cells remain growth arrested, the more time they require to enter S phase. This prolongation of the prereplicative phase has been localized to a point early in G₁, after the induction of “immediate early” G₁ genes such as c-fos and c-jun but before maximal expression of “early” G₁ genes such as ornithine decarboxylase (ODC). Understanding the molecular basis for ODC mRNA induction can therefore provide information about the molecular events which regulate the progression of cells out of long-term quiescence into G₁ and subsequently into DNA synthesis. Studies utilizing electrophoretic mobility shift assays (EMSA) of nuclear extracts from short- and long-term quiescent WI-38 cells identified a region of the human ODC promoter at ?491 bp to ?474 bp which exhibited a protein binding pattern that correlated with the temporal pattern of ODC mRNA expression. The presence of a CACGTG element within this fragment, studies with antibodies against c-Myc and Max, the use of purified recombinant c-Myc protein in the mobility shift assay, and antisense studies suggest that these proteins can specifically bind this portion of the human ODC promoter in a manner consistent with growth-associated modulation of the expression of ODC and other early G₁ genes following prolonged quiescence. These studies suggest a role for the c-Myc/Max protein complex in regulating events involved in the progression of cells out of long-term quiescence into G₁ and subsequently into S. © 1995 Wiley-Liss, Inc.     

Autophagy is required for G1/G0 quiescence in response to nitrogen starvation in Saccharomyces cerevisiae

In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G₀, autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.     

Autophagy is required for G1/G0 quiescence in response to nitrogen starvation in Saccharomyces cerevisiae

In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G₀, autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.     

Synchronization of Cultured Vascular Smooth Muscle Cells Following Reversal of Quiescence Induced by Treatment with the AntioxidantN-Acetylcysteine

Smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. Recently we demonstrated the thiol antioxidantN-acetylcysteine (NAC) inhibits constitutive NF-κB/Rel activity and growth of vascular SMCs. Here we show that treatment of human and bovine aortic SMC with the thiol antioxidant NAC causes cells to exit the cell cycle and remain quiescent as determined by a greatly reduced incorporation of [³H]thymidine and G₀/G₁DNA content. Removal of NAC from the culture medium stimulates SMCs to synchronously reenter the cell cycle as judged by induction of cyclin D1 and B-mybgene expression during mid and late G₁phase, respectively, and induction of histone gene expression and [³H]thymidine incorporation during S phase. The time course of cyclin D1, B-myb,and histone gene expression after NAC removal was similar to that of serum-deprived cells induced to resume cell cycle progression by the addition of fetal bovine serum to the culture medium. Taken together, these results indicate that NAC treatment causes SMCs to enter a reversible G₀quiescent, growth-arrested state. Thus, NAC provides an important new method for synchronizing SMCs in culture.     

SPECIFIC CHALONE INHIBITION OF THE REGENERATION OF THE JB-1 ASCITES TUMOUR STUDIED BY FLOW MICROFLUOROMETRY

The variation in the DNA distribution in the JB-1 and the Lla₂ ascites tumour was investigated by means of flow microfluorometry (FMF) in the plateau stage and during the initiation of the regeneratave growth induced by percutaneous aspiration. The study showed that a considerable influx of cells with G₁ DNA content into the S phase occurred in both tumours about 10 hr after aspiration. In the JB-1 tumour, these initial regenerative changes could be reversibly blocked by injections of cell-free plateau JB-1 ascitic fluid or an ultrafiltrate of this ascites. In contrast to these observations no delay in the regenerative changes was observed in the Lla₂ tumour after treatment with JB-1 ascites or the ultrafiltrate. The study supports the assumption of a specific growth regulation of the JB-1 ascites tumour and emphasizes the suitability of FMF analyses in cell-kinetic studies in which short-term fluctuations take place in the distribution of cells with different DNA content.     

EXISTENCE OF TWO CHALONE-LIKE SUBSTANCES IN INTESTINAL EXTRACT FROM THE ADULT NEWT, INHIBITING EMBRYONIC INTESTINAL CELL PROLIFERATION

The inhibiting effect of tissue extract from fully differentiated intestinal mucosa of adult animals on proliferation kinetics of exponentially growing embryonic epithelial gut cell populations was studied in the newt Pleurodeles waltlii. Crude extract was fractionated by G-200 Sephadex chromatography and the effect of fractions on cell proliferation was studied using both mitotic index and ³H-thymidine incorporation methods. The inhibitions we obtained were then displayed by means of cytophotometric study of age distribution of intestinal gut cells around the cell cycle, measuring the Feulgen-DNA content. The results revealed the presence of two chalone-like substances in the intestine of adults. One (factor 1) is characterized by a molecular weight of between 120,000 and 150,000 and inhibits the cell cycle at the end of the G₁ phase, the other (factor 2) is characterized by a molecular weight lower than 2000 and inhibits the cell cycle in the course of the G₂ phase. The cells delayed in the G₂ phase escape from inhibition but the cells delayed in the G₁ phase do not, although availability time of both factor 1 and factor 2 is about 12 hr. It is thus thought that cells prevented from dividing in G₁ phase are indefinitely delayed in this phase and possibly differentiate.     

Image analysis of in situ cell cycle related changes of PCNA and Ki-67 proliferating antigen expression

Abstract. This study reports on the proliferating cell nuclear antigen (PCNA) and Ki-67 cell cycle related expression and distribution pattern analysed in the same cells. MCF-7 cells were synchronized by mitotic detachment and triple stained for DNA, PCNA and Ki-67. The major cell type was identified on each time sample as a function of the PCNA/Ki-67 pattern, and both antigens as well as DNA were quantified. During G₁ phase, the expression of PCNA greatly increased whereas Ki-67 content decreased. During S phase, nuclear Ki-67 content continuously increased especially in the second half of this phase, mainly due to the accumulation of the antigen in the nucleoli. During G₂ phase, the antigen significantly passed into the nucleoplasm, its content continued to increase and reached its maximum in mitotic cells. Nuclear PCNA content mostly increased in the first part of S phase and sharply declined in mitotic cells as the antigen shifted to the cytoplasm. Cells showing PCNA positive Ki-67 negative labelling were observed in all time samples from the beginning of the experiment. Their nuclear size, DNA content (of G₁ cells), PCNA content (equivalent to the content of some late G, cells) and time occurrence (their percentage increased after the last late G₁ cells had disappeared) tend to indicate that these cells have left the cycle by the end of G₁ phase to enter a quiescent state. Cells coming out of mitosis split into two groups according to their Ki-67/PCNA content. The biggest fraction was PCNA negative and Ki-67 positive while the smallest showed positive staining for both antibodies. Cells of this second cohort slowly lost their 1–67 while their PCNA content increased as they moved through G₁. Concurrently, most of the cells of the first cohort (here called Q2 and Q3 cell types) lost their Ki-67 without increasing their PCNA content; then they joined cells of the second cohort by increasing their PCNA content at the end of G, phase. Some cells of this first cohort can also increase their PCNA and thus reach cells of the first cohort before the end of G₁ phase. The existence of these two main cell cohorts suggests that cells after mitosis differ in some way that make them progress dlfferently through G₁. Some cells seem to go through early G₁ (G_1a and late G₁ (G_lb) while others may come out of mitosis committed to go through the following cycle by directly entering late G₁ compartment.     

Tumor-specific cell-cycle decoy by Salmonella typhimurium A1-R combined with tumor-selective cell-cycle trap by methioninase overcome tumor intrinsic chemoresistance as visualized by FUCCI imaging

We previously reported real-time monitoring of cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G₀/G₁ phase. Longitudinal real-time FUCCI imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, and had little effect on the quiescent cancer cells. Resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Thus cytotoxic chemotherapy which targets cells in S/G₂/M, is mostly ineffective on solid tumors, but causes toxic side effects on tissues with high fractions of cycling cells, such as hair follicles, bone marrow and the intestinal lining. We have termed this phenomenon tumor intrinsic chemoresistance (TIC). We previously demonstrated that tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) decoyed quiescent cancer cells in tumors to cycle from G₀/G₁ to S/G₂/M demonstrated by FUCCI imaging. We have also previously shown that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G₂, shown by cell sorting as well as by FUCCI. In the present study, we show that sequential treatment of FUCCI-expressing stomach cancer MKN45 in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle, with subsequent rMETase to selectively trap the decoyed cancer cells in S/G₂ phase, followed by cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy to kill the decoyed and trapped cancer cells completely prevented or regressed tumor growth. These results demonstrate the effectiveness of the praradigm of “decoy, trap and shoot” chemotherapy.