Distribution of Interdivisional Times In Proliferating and Differentiating Friend Murine Erythroleukaemia Cells

Abstract. The interdivisional times of Friend murine erythroleukaemia cells which are growing continuously, or during terminal erythroid differentiation after exposure to dimethyl sulphoxide (DMSO), were determined by time lapse video photography. the median interdivisional times were found to increase from 11.75 hr before exposure to DMSO, to 24.0 hr at 72 hr after exposure. This increase in median interdivisional time was accompanied by an increase in heterogeneity of interdivisional times (% CV = 8-5 → 40.8), by an increase in the similarity of sister interdivisional times (r_yy= 0.622 → 0.925), and by a decrease in the fraction of cells observed to divide ( F = 1. 0 → 0.807). Cells exposed to DMSO for 72 hr can be induced to divide at least once with nearly normal interdivisional times, if they are resuspended at a tenfold higher cell concentration. Computer simulations of cell cycle regulation, based on the opposing reactions model of Murphy, generate interdivisional time distributions which resemble the experimental data better than the single transition probability model of Smith and Martin.     

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells during their erythroid differentiation

The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post-exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis by demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin.     

Cryopreservation of immature porcine testis tissue to maintain its developmental potential after xenografting into recipient mice

The purpose of this study was to develop effective strategies for cooling and cryopreservation of immature porcine testis tissue that maintain its developmental potential. Testes from 1-wk-old piglets (Sus domestica) were subjected to 1 of 12 cooling/cryopreservation protocols: as intact testes, cooling at 4 °C for 24, 48, or 72 h (Experiment 1); as fragments, programmed slow-freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol (Experiment 2); or solid-surface vitrification using DMSO, glycerol, or ethylene glycol, each using 5-, 15-, or 30-min cryoprotectant exposure times (Experiment 3). For testis tissue xenografting, four immunodeficient recipient mice were assigned to each protocol, and each mouse received eight grafts. Recipient mice were killed 16 wk after grafting to assess the status of graft development. Based on morphology and in vitro assessment of cell viability, cooling of testis tissue for up to 72 h maintained structural integrity, cell viability, in vivo growth, and developmental potential up to complete spermatogenesis comparable with that of fresh tissue (control). In frozen-thawed testis tissues, higher numbers of viable cells were present after programmed slow-freezing using glycerol compared with that after DMSO or ethylene glycol (P < 0.001). Among the vitrified groups, exposure to DMSO for 5 min yielded numerically higher viable cell numbers than that of other groups. Cryopreserved tissue fragments recovered after xenografting had normal spermatogenesis; germ cells advanced to round and elongated spermatids after programmed slow-freezing using glycerol, as well as after vitrification using glycerol with 5- or 15-min exposures, or using DMSO for a 5-min exposure.     

Consequences of parental exposure to epidermal growth factor for progeny cell division

The consequences of parental exposure to epidermal growth factor (EGF), for progeny cell cycle times was investigated. Slowly dividing mouse 3T3 fibroblasts were exposed to EGF for 8 hr, the EGF was withdrawn, and the cell cycle times of parental and progeny cells were measured by time-lapse video microscopy. It was observed that exposure to EGF induced a round of cell division following a lag phase of approximately 8 hr. The progeny of these cells exhibited accelerated cell cycle times compared to cells that had not been exposed to EGF. Parental cell division time was significantly correlated with progeny cell cycle time. Sibling progeny cell cycle times were also significantly correlated. EGF can therefore apparently exert an effect on the cell cycle times of more than one generation of cells.     

Studies on the effect of experimental inflammation on sialyltransferase in the mouse and guinea pig

The effect of inflammation on sialyltransferase was studied in the mouse and guinea pig. There was a three-fold increase in mouse liver sialyltransferase activity reaching a maximum at 72 hr after inflammation; serum levels were increased by five-fold at 72 hr after inflammation. The response of guinea pig sialyltransferase was slower and of lower magnitude compared with the response of the mouse enzyme; liver and serum sialyltransferase increased by about 50% reaching a maximum at 96 hr after inflammation. The specificity of the enzyme that responded to inflammation in the mouse and guinea pig was found to be Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase, the same enzyme activity that was shown to be an acute phase reactant in earlier studies in the rat (Kaplan et al., 1983).     

Salinity stress results in rapid cell cycle changes of tilapia (Oreochromis mossambicus) gill epithelial cells

We have developed a technique for immunocytochemistry of fish gill cells that we used to quantify tilapia (Oreochromis mossambicus) mitochondria-rich cells (MRC) and other gill cells (non-MRC) within different cell cycle phases by laser scanning cytometry. Gill cells fixed on coverslips were triple stained with propidium iodide to distinguish G1 vs. G2 phases, Ser10-phosphorylated histone H3 antibody to label mitotic cells, and Na(+)/K(+) ATPase antibody to label MRC. These parameters were measured at 0 (control), 4, 8, 16, 24, 48, 72, and 168 hr (1 week) following exposure of freshwater (FW) acclimated fish to 2/3 seawater (SW). MRC increased mitotic activity very rapidly peaking at 8 hr following SW exposure. This change in mitotic MRC is indicative of epithelial reorganization during SW acclimation. In contrast to MRC, the proportion of non-MRC (likely pavement cells (PVC)) in mitosis did not change significantly in response to SW exposure. Moreover, twice as many MRC were in mitosis compared with non-MRC, suggesting that MRC turn over faster than other cell types during SW acclimation. Following the mitosis peak, MRC accumulated in G2 phase over a period of 16-72 hr post-SW exposure. We also observed G2 arrest with similar kinetics following SW exposure in tilapia non-MRC (likely PVC). We interpret the G2 arrest that occurs after an initial wave of transient increase in MRC mitosis as a means for conserving energy for dealing with the osmotic stress imposed during the exposure of FW fish to SW.     

Src-MAPK信号通路参与透明质酸寡糖片段诱导PIEC细胞的增殖

探讨透明质酸小片段(Hyaluronan oligosaccharides,o-HA)对血管内皮细胞生长的影响及机制。采用细胞计数、流式细胞术、Western blot等方法,检测o-HA作用于内皮细胞后,Src激酶、丝裂原活化蛋白激酶MAPK(ERK-1/2)的磷酸化程度及细胞周期蛋白D1的表达水平。o-HA在10μg/ml浓度时可明显地促进血管内皮细胞的增殖,包括细胞周期水平及细胞数量。继续增加o-HA的浓度及延长培养时间均未出现进一步的变化;细胞增殖在12h开始出现,72h达到高峰。1μg/ml的o-HA作用于细胞2min后,胞内Src激酶、ERK-1/2的磷酸化程度即增强,并在3h后检测到周期蛋白D1的表达水平增高。o-HA具有促进血管内皮细胞增殖的作用,其机制可能是通过激活Src激酶及细胞内MAPK信号通路,从而促进血管内皮细胞生长;该作用也可能与上调周期蛋白D1的表达有关。     

Analysis of the growth kinetics of a human lymphoma cell line

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 X 10(4)--5 X 10(5) cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with 3H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. The average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. The generation time determined by PLM was 31.1 hr with a SD of 4.7 hrs. Transit times for each stage were: TG1 = 10.6 hr, TS = 9.9 hr, TG2 = 9.9 hr, and TM = 0.7 hr. Repeated experiments using continuous labeling with 3H-TdR demonstrated a TG2 of 6.3 hr. The longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give TG1 = 14.1 hr, TS = 9.9 hr, TG2 = 6.3 hr, and TM = 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. The average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

Studies on the effect of the hepatocyte-stimulating factor on galactose-beta 1----4-N-acetylglucosamine alpha 2----6-sialyltransferase in cultured hepatocytes

Rat hepatic Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase is released into the blood at elevated levels following an inflammatory challenge: this is a typical response of the group of plasma proteins known as acute-phase reactants. In the present study, primary cultures of liver parenchymal cells are used to demonstrate that the same hepatic cell type that produces plasma proteins such as fibrinogen also produces and releases sialyltransferase. Hepatic production of sialyltransferase is stimulated by a major regulator of hepatic acute-phase reactant production, the hepatocyte-stimulating factor (HSF), while another monokine, interleukin-1, does not affect hepatocyte sialyltransferase production. The maximum increase in sialyltransferase occurs 48 h after exposure to HSF which is considerably later than the fibrinogen response. The sialyltransferase that is stimulated by HSF is the Gal beta 1----4GlcNAc alpha 2----6 isozyme.     

Growth kinetics of cells following freezing in liquid nitrogen

The growth kinetics of cells frozen to ?196 °C were monitored after thawing by various techniques. Progression through the cell cycle in the exposed generation was observed by monitoring cell growth either via multiplicity counts or by electronic cell counts of trypsinized suspensions. Subsequent generations were followed by time-lapse microcinematography.The division delay in the exposed generation of exponential-phase cells was dependent on cell age at the time of freezing and varied from 4 to 8 hr. The time of the first generation was still prolonged significantly but subsequent generations revealed cell cycle times that are comparable to unfrozen cells. In the case of plateau-phase cells, mitosis was delayed 7 hr in the exposed generation. This is 50% longer than the delay seen for pre-DNA synthetic g₁ cells in exponentially growing cultures.A rather important observation in this study was that frozen-thawed cells which divide once will probably continue dividing whereas eventual nonsurvivors are not likely to divide at all. The latter, however, remain active for more than 35 hr as observed microscopically, hence possibly indicating residual metabolic activity.     

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.     

Analysis of population cytokinetics of chick myocardial cells in tissue culture

The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.     

Murine mammary tumour cells in vitro. II. Recruitment of quiescent cells

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 approximately equal to 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. Since 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.     

Ultrastructural analysis of acute immune thrombocytopenia in mice: dissociation between alterations in megakaryocytes and platelets

Thrombopoiesis was studied in mice after the induction of acute immune thrombocytopenia with platelet antiserum (PAS). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 4, 8, 12, 24, 48, 72, and 120 hr after administration of PAS. Four to 24 hr after injection of PAS, the majority of bone marrow MK were normal in size and organelle distribution. The demarcation membrane system (DMS) extended normally throughout the mature cell cytoplasm at these times. However, approximately 50% of MK observed 48 hr and 72 hr after injection of PAS were significantly larger than normal, and often had demarcation membranes confined to an area between a peripheral organelle-deficient zone and a central nuclear zone. The median values for sectional areas of platelets obtained 8-72 hr after administration of PAS were significantly greater than the median value for sectional areas of platelets in a pooled control sample. The proportion of cytoplasm to surface-connected canalicular system appeared greater than normal in most large platelets from the PAS samples; and increased numbers of profiles of Golgi complex and endoplasmic reticulum were observed. By 48 hr post-injection of PAS (at which time the modal ploidy class of MK has shifted from 16N to 32N; Corash et al., Blood 70:177, 1987), most platelets were normal in size and cytoplasmic appearance. At 120 hr post-injection of PAS, virtually all platelets exhibited a normal size and complement of organelles, and MK also had returned to normal. Our data indicate that in response to acute thrombocytopenia, MK prematurely release platelets which differ from normal platelets in size and cytoplasmic appearance. There was a marked dissociation between alterations in platelets and MK, since a statistically significant increase in platelet sectional area occurred 40 hr before the shift in modal ploidy class of MK, and platelet size subsequently decreased toward normal during the period that has been shown to be associated with the maximum shift in MK ploidy. These results strongly suggest that the characteristics of platelet release do not depend on the ploidy or cytoplasmic characteristics of MK.     

Characterizing the mechanism for ginsenoside-induced cytotoxicity in cultured leukemia (THP-1) cells

Pure ginsenoside standards (saponins Rh2, PD, and PT), along with an Rh2-enhanced North American ginseng (Panax quinquefolius) leaf extract (LFRh2), were tested for cytotoxic activity in cultured THP-1 leukemia cells. Thermal treatment of ginseng leaf resulted in production of both Rh2 and Rg3 content that was confirmed by liquid chromatography - mass spectrometry (LC-MS). Flow cytometry of cells stained with annexin V - fluorescein isothiocyanate and propidium iodide showed that the LFRh2 significantly (p < or = 0.05) increased apoptosis (18% +/- 0.4%) after 23 h at a concentration that inhibited cell viability by 50% (LC50 (72 h) = 52 microg/mL. In comparison, a similar significant (p < or = 0.05) increase in apoptotic cell numbers occurred at 41 h of exposure for pure ginsenoside standards, PD (LC50 (72 h) = 13 microg/mL), PT (LC50 (72 h) = 19 microg/mL), and Rh2 (LC50 (72 h) = 15 microg/mL). Although no further increase in apoptosis was observed in THP-1 cells after exposure to increasing concentrations of LFRh2 and pure Rh2, PD, and PT standards, a significant (p < 0.05) increase in the percentage of necrotic cells did occur after exposure of cells to different ginsenosides at elevated concentrations. THP-1 caspase-3 activity was greatest (p 相似文献   

Memory of MEL cells to a previous exposure to inducer.

The mechanism of commitment of murine erythroleukemia (MEL) cells to terminal differentiation has been examined. Before a significant proportion of cells becomes committed, a lag period of at least 9 hr of exposure to inducer is observed. Cells withdrawn from inducer can reinitiate commitment without a lag when reexposed. The proportion of committed cells in a culture discontinuously exposed to inducer is identical to that in a continuously exposed culture even if withdrawal from inducer lasts for 18 hr. The ability to tolerate an interruption in the exposure has been termed "memory." The memory of a previous exposure to inducer is complete up to 18 hr. It is partially erased after 36 hr and completely erased after 72 hr. The length of time the memory persists is not affected by the length of the initial exposure to inducer. These results suggest that a cellular component necessary for the commitment event accumulates in response to inducer and that this component has a decay time on the order of 10 hr.     

The differential effects of calcium starvation on Duchenne muscular dystrophy fibroblasts

The effects of nutrient deprivation on normal and Duchenne muscular dystrophy fibroblasts were examined. The requirements for Ca2+ and fetal bovine serum were assessed by their effects on the cells' ability to support viral replication, and by ability of the cells to divide in the presence of low levels of these nutrients. When grown in Ca2+-deficient media, Duchenne fibroblasts supported viral replication at a rate 2- to 2.5-fold greater than did normal fibroblasts. At normal Ca2+ levels, Duchenne fibroblasts supported viral replication at levels slightly lower than their normal counterparts. After 48 hr in medium containing 0.2 mM Ca2+, the growth of normal cells was arrested, while Duchenne fibroblasts were relatively unaffected. When grown in medium containing either 0.2 or 2.0% serum, the growth of normal cells was arrested within 48 hr, with cell death occurring within 72 hr. Duchenne fibroblasts continued to divide at these serum levels for 72 hr, reaching higher cell densities than normal cells. These results suggest that a defect related to Ca2+ metabolism may be part of the Duchenne phenotype, which could be used to identify Duchenne muscular dystrophy cells.     

Physiological oxygen tension modulates the chemically induced mitogenic response of cultured rat hepatocytes

Freshly isolated rat hepatocytes were cultured at periportal- (13% O₂) or perivenous-like (4% O₂) oxygen tension and exposed to subtoxic exposure levels of cyproterone acetate (CPA: 10–330 μM), phenobarbital (PB: 0.75-6 mM), and dimethylsulfoxide (DMSO: 0.1–3.3%) from 24–72 h after seeding. Induced alterations in ploidy, in the number of S-phase cells, the degree of binuclearity, and cellular protein content were determined by twin parameter protein/DNA flow cytometry analysis of intact cells and isolated nuclei. CPA and PB increased whereas DMSO decreased dose dependently the total number of S-phase cells. The changes differed within individual ploidy classes and were modulated by the oxygen tension. CPA increased and DMSO decreased the number of S-phase cells preferentially among the diploid hepatocytes at periportal-like oxygen tension. In contrast, PB increased binuclearity and S-phase cells mainly among the tetraploid hepatocytes at perivenous-like oxygen tension. Cellular protein content increased dose dependently after exposure to the hepatomitogens (CPA, PB) and decreased after exposure to DMSO at both oxygen tensions. Comparison with in vitro data proves that chemicals which interact with cells from the progenitor liver compartment (CPA, DMSO) exert their mitogenic activity best in cultures at periportal-like oxygen tension preferentially in diploid hepatocytes, whereas chemicals which affect cells from the functional compartment show a higher activity at perivenous-like oxygen tension. Physiological oxygen tension seems to be an effective modulator of the proliferative response of cultured rat hepatocytes similar to that expected for periportally or perivenously derived hepatocytes. © 1993 Wiley-Liss, Inc.     

Interactions of dimethyl sulfoxide and granulocyte-macrophage colony-stimulating factor on the cell cycle kinetics and phosphoproteins of G1-enriched HL-60 cells: evidence of early effects on lamin B phosphorylation

We have found that GM-CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL-60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL-60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombinant human GM-CSF and/or 0-1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]-orthophosphate-labeled cell proteins separated by giant 2-D gel electrophoresis. Results were correlated with 1) intracellular pH, determined by measurement of BCECF fluorescence; 2) [32P]-orthophosphate uptake; 3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide-stained cells; and 4) growth, determined by cell volume and concentration. GM-CSF stimulated and DMSO inhibited the GM-CSF-stimulated phosphorylation of 1 protein (approximately 65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1, not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15-30 minutes), cell volume change (16-24 hr), and cell concentration increase (28-32 hr). GM-CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synchronization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM-CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM-CSF-DMSO antagonism is exerted through the same mitogenic pathway, that a major locus of cytokinetic effect is on G1 to S transit, and that nuclear envelope protein phosphorylation is an important early event.     

Changes in the ultrastructure and DNA and protein content in human atrial myocytes in cardiac hyperfunction due to mitral valve defects

Biopsies from human right auricles were obtained during open heart surgery, prior to valve replacement, from six patients (aged from 20 to 49 years) with rheumatic heart disease. DNA and the total protein contents were measured in isolated myocytes by means of the two wave-length scanning cytophotometry after the double Feulgen and Naphthol yellow S staining procedure. In all the biopsies polyploid hypertrophied myocytes predominate. The hypertrophic, nondegenerated cells and the cells with degenerative changes of varying severity (in the first place, changes of contractile apparatus and membranes) are present. The highest degree of cell ploidy occurs in patients of functional class IV according to the New York Heart Association classification, 72 to 98% of cells displaying octaploid and higher DNA values. With the increase in ploidy of myocytes in series 2c----4c----8c----16c----32c----64c the protein content increases only as 2.0----3.0----5.8----7.8----13.0----16.8. Neither direct correlation between the ploidy level and the degree of cell degeneration, no inverse correlation between the degree of degeneration and the value of ejection fraction was observed.