Myocyte polyploidy in human cardiac pathology]

With general atherosclerosis, the ploidy of left ventricle myocytes in the hearts of patients that underwent infarction corresponds to the norm variation irrespective of the ventricle and heart weights. At heart diseases the myocyte nucleus ploidy is often much higher than the norm variability both in hypertrophied ventricles and in those with normal weight. An additional polyploidization is suggested that may occur at some natural ontogenetic periods of human development (in the childhood) during heart diseases both innate or spontaneously appearing at the particular time. Unlike, the myocardial hypertrophy in adults does not stimulate myocyte polyploidy.     

Levels of DNA and sex chromatin bodies in the myocyte nuclei of different sections of the human heart

Nuclei of ventricular, atrial and atrioventricular node myocytes of normal and hypertrophied human heart were studied on squash preparations and on 12 micron sections after the Feulgen staining. The cytophotometric DNA measurements have shown a distinction in the degree of polyploidization of nuclei in different heart compartments. In contrast to ventricular and atrial myocardia, in which polyploid nuclei predominate, the conduction system myocytes contain 77-88% of diploid nuclei. A correlation between DNA content and the number of sex chromatin bodies was observed for myocyte nuclei from all the compartments under investigation.     

Methodological aspects of flow cytometric analysis of DNA polyploidy in human heart tissue

Summary The purpose of the study was to investigate the possibilities of flow cytometry (FCM) for the analysis of DNA polyploidy in human heart tissue. Suspensions of single nuclei were prepared with the detergenttrypsin procedure and stained with propidium iodide. A mathematical correction procedure was developed to correct for background and clumping. For diploid model populations of chicken and trout red blood cells this correction reduced artifactual fractions in the FCM DNA profile to less than 0.5%, indicating that interference by background and clumping was almost completely overcome by this correction procedure. FCM DNA profiles were obtained from 12 hypertrophic and 7 normal human adult hearts. Clear differences were found between the DNA profiles from the normal and the hypertrophic hearts, the latter showing a higher degree of polyploidization. From the corrected DNA profiles, six different polyploidization parameters were computed, all of which showed a significant correlation with at least three out of four different parameters for heart hypertrophy. FCM appears to be a reliable method for the measurement of polyploidization in heart tissue, provided background and clumping are corrected for.In honour of Prof. P. van Duijn     

Methodological aspects of flow cytometric analysis of DNA polyploidy in human heart tissue

The purpose of the study was to investigate the possibilities of flow cytometry (FCM) for the analysis of DNA polyploidy in human heart tissue. Suspensions of single nuclei were prepared with the detergent-trypsin procedure and stained with propidium iodide. A mathematical correction procedure was developed to correct for background and clumping. For diploid model populations of chicken and trout red blood cells this correction reduced artifactual fractions in the FCM DNA profile to less than 0.5%, indicating that interference by background and clumping was almost completely overcome by this correction procedure. FCM DNA profiles were obtained from 12 hypertrophic and 7 normal human adult hearts. Clear differences were found between DNA profiles from the normal and the hypertrophic hearts, the latter showing a higher degree of polyploidization. From the corrected DNA profiles, six different polyploidization parameters were computed, all of which showed a significant correlation with at least three out of four different parameters for heart hypertrophy. FCM appears to be a reliable method for the measurement of polyploidization in heart tissue, provided background and clumping are corrected for.     

Polyploidy: significance for cardiomyocyte function and heart aerobic capacity

Somatic polyploidy, defined as genome multiplication, was found in all differentiated mammalian tissues. The highest level of such a polyploidy was found in the myocardium. This phenomenon was shown to be associated with changes in the pattern of gene expression. Hence, polyploidization may create cells with new physiology. The effect of polyploidy on the heart function has never been studied. The aim of the present study was to investigate the effect of polyploidy on cardiomyocyte functioning and heart aerobic capacity. DNA and the total protein content, nucleolar activity reflecting the rate of rRNA synthesis and, consequently, ribosome biogenesis, were measured in ventricular myocytes isolated from the human and from 21 mammalian species by image cytometry and microscopic morphometry. The total protein content was estimated after staining slides with naphtol-yellow dye. For measurement of DNA and nucleolar area, staining with Hoechst and AgNO3 was applied. Cardiac aerobic capacity was evaluated by the heart mass to body mass ratio. A negative correlation between the heart index and the average cell ploidy was revealed (r = -0.79; P < 0.0001). The average genome number per myocyte was registered to be higher by approximately 35% in the sedentary mammals, with the heart index about 0.4% from body mass, than in the athletes with heart index about 0.6% of body mass. Polyploidization was shown to be associated with a sharp decrease in the protein/DNA ratio in cardiomyocytes. As a result, cardiomyocytes in the athletic mammals with poorly polyploid hearts have much higher protein content per genome than do cells in the sedentary species with highly polyploid hearts. Surprisingly, despite decreased protein/DNA ratio, the nucleolar area per genome significantly increased with polyploidization, indicating the imbalance between the cellular protein content and the rate of ribosome biogenesis. Such an imbalance should obviously impair cardiac function, because the additional genomes take some valuable space and biological resources from the cell, which could have been otherwise directed to the maintenance of cardiomyocyte contractile machinery. It is generally accepted that somatic polyploidy is associated with oxidative stress and energetic starvation. Thus, we suppose that additional genomes may serve for cardiomyocyte protection from oxidative damage in the hearts.     

The capacity for reactive DNA synthesis of the myocytes in the heart conduction system in experimental and clinical myocardial pathology]

The DNA synthesis has been studied in the conductive system (CS) myocytes, compared to that in atrial and ventricular myocytes: 1) in the left ventricular myocardial infarction induced in two- and three-week-old and adult rats, 2) after isoproterenol injections to adult rats and mice, and 3) in the hypertrophied human heart. The extent of DNA synthesis reactivation was evaluated by the cumulative labeling indices in experiments with multiple 3HTdR injections to rats and mice. In the human cardiac myocyte nuclei, the DNA content was determined by the Feulgen-cytophotometry. The difference between the control and experimental mean values of the labeling indices for CS myocyte nuclei was statistically significant only for atrioventricular part of the CS in the infarcted hearts of adult rats. In the human heart CS the ability of myocytes to polyploidization varies from one cell type to another, the lowest being in nodal cells.     

Nuclear size of myocardial cells in end-stage cardiomyopathies

OBJECTIVE: To determine the alteration of nuclear size in myocardial cells and the relationship between nuclear size and DNA ploidy classes in normal and cardiomyopathic human hearts. STUDY DESIGN: The study group consisted of 46 hearts obtained at biopsy. These patients had undergone cardiac transplantation for intractable congestive heart failure (18 cases with ischemic cardiomyopathy and 28 cases with idiopathic dilated cardiomyopathy). Another 10 hearts were collected at autopsy and used as control hearts according to preautopsy, autopsy and histology criteria. One hundred fibroblasts and 200 myocytes were evaluated in each ventricle. The nuclear area and DNA content were estimated using image cytometry. RESULTS: End-stage ischemic and dilated cardiomyopathies were characterized by an increase in nuclear size of both the myocyte and nonmyocyte population. The nuclear area of interstitial cells increased about 30% in cardiomyopathic hearts. Augmentation of average nuclear area of myocytes was 1.2-fold in the ischemic group and about 1.5-fold in the dilated group as compared with the control group. Also, a tendency was found for the coefficient of variation of average nuclear area to decrease in the interstitial cell population and increased in the myocyte population in cardiomyopathic situations. Furthermore, the nuclear area of myocytes enlarged as augmentation of nuclear DNA content. The relative nuclear areas of myocytes can be presented as: 2c:4c:8c:16c :32c:64c = 1:1.65:2.75:4.60:7.25:9.18. CONCLUSION: The increase in nuclear size follows either one of two different processes: the first does not involve an increase in DNA content, whereas the second is concomitant with an incremental increase in DNA content. In the first instance, the enlargement of nuclear size is limited. In the second, augmentation of nuclear size can become very impressive. In end-stage ischemic and dilated cardiomyopathies, the nuclear growth of myocytes and interstitial cells may be due to different mechanisms. Enlargement of the nuclear area of myocytes represents a complex process, including simple nuclear hypertrophy, polyploidization and multinucleation. The main pattern of nuclear growth of interstitial cells is nuclear hypertrophy without an increase in DNA content.     

Different response of cellular DNA content to cardiac hypertrophy in human and rat heart myocytes

1. Rat and human heart myocytes adapt to overload-induced hypertrophy differently. 2. Human myocyte nuclei respond with polyploidization and multinucleation, thus increasing the DNA content per myocyte from 20 to 40 pg. As a result, nuclear DNA content per 10,000 microns3 of cell volume decreases from 12 to 10 pg. 3. In rat hearts with aortic constriction nuclear DNA content remains constant (13 pg), and the DNA content per 10,000 microns3 of myocyte volume falls from 9 to 6 pg. 4. We hypothesize that "dilution" of nuclear DNA in the hypertrophied rat heart myocyte limits the capacity to hypertrophy (much less than 100%). 5. The human heart myocyte, which is able to compensate for dilution of nuclear DNA, may increase in size more than three-fold. 6. The lower limit of DNA content per unit of myocyte volume is 6 pg/10,000 microns3 in both species.     

Decrease in the density of t-tubular L-type Ca2+ channel currents in failing ventricular myocytes

In some forms of cardiac hypertrophy and failure, the gain of Ca(2+)-induced Ca(2+) release [CICR; i.e., the amount of Ca(2+) released from the sarcoplasmic reticulum normalized to Ca(2+) influx through L-type Ca(2+) channels (LTCCs)] decreases despite the normal whole cell LTCC current density, ryanodine receptor number, and sarcoplasmic reticulum Ca(2+) content. This decrease in CICR gain has been proposed to arise from a change in dyad architecture or derangement of the t-tubular (TT) structure. However, the activity of surface sarcolemmal LTCCs has been reported to increase despite the unaltered whole cell LTCC current density in failing human ventricular myocytes, indicating that the "decreased CICR gain" may reflect a decrease in the TT LTCC current density in heart failure. Thus, we analyzed LTCC currents of failing ventricular myocytes of mice chronically treated with isoproterenol (Iso). Although Iso-treated mice exhibited intact t-tubules and normal LTCC subunit expression, acute occlusion of t-tubules of isolated ventricular myocytes with osmotic shock (detubulation) revealed that the TT LTCC current density was halved in Iso-treated versus control myocytes. Pharmacological analysis indicated that kinases other than PKA or Ca(2+)/calmodulin-dependent protein kinase II insufficiently activated, whereas protein phosphatase 1/2A excessively suppressed, TT LTCCs in Iso-treated versus control myocytes. These results indicate that excessive β-adrenergic stimulation causes the decrease in TT LTCC current density by altering the regulation of TT LTCCs by protein kinases and phosphatases in heart failure. This phenomenon might underlie the decreased CICR gain in heart failure.     

Importance of antioxidant and antiapoptotic effects of beta-receptor blockers in heart failure therapy

The present study was carried out to determine whether beneficial effects of carvedilol in congestive heart failure (CHF) are mediated via its beta-adrenergic blocking, antioxidant, and/or alpha-adrenergic blocking action. Rabbits with heart failure induced by rapid cardiac pacing were randomized to receive subcutaneous carvedilol, metoprolol, propranolol plus doxazosin, or placebo pellets for 8 wk and compared with sham-operated rabbits without pacing. We found rapid cardiac pacing produced clinical heart failure, left ventricular dilation, and decline of left ventricular fractional shortening. This was associated with an increase in left ventricular end-diastolic pressure, decrease in left ventricular first derivative of left ventricular pressure, and myocyte hypertrophy. Tissue oxidative stress measured by GSH/GSSG was increased in the heart with increased oxidation product of mitochondrial DNA, 8-oxo-7,8-dihydro-2'-deoxyguanosine, increase of Bax, decrease of Bcl-2, and increase of apoptotic myocytes as measured by anti-single-stranded DNA monoclonal antibody. Administration of carvedilol and metoprolol, which had no effect in sham animals, attenuated cardiac ventricular remodeling, cardiac hypertrophy, oxidative stress, and myocyte apoptosis in CHF. In contrast, propranolol plus doxazosin, which has less antioxidant effects, produced smaller effects on left ventricular function and myocyte apoptosis. In all animals, GSH/GSSG correlated significantly with changes of left ventricular end-diastolic dimension (r = -0.678, P < 0.0001), fractional shortening (r = 0.706, P < 0.0001), and apoptotic myocytes (r = -0.473, P = 0.0001). Thus our findings suggest antioxidant and antiapoptotic actions of carvedilol and metoprolol are important determinants of clinical beneficial effects of beta-receptors in the treatment of CHF.     

Upregulation of redox-regulating protein,thioredoxin, in endomyocardial biopsy samples of patients with myocarditis and cardiomyopathies

An important role of redox regulation in myocardial diseases and heart failure has been postulated. Thioredoxin (TRX) is a redox-regulating protein. Recent studies indicated a possible association between plasma TRX concentrations and the severity of heart failure. Accordingly, we investigated the myocardial expression of TRX in patients with myocarditis and cardiomyopathies. Four cases of hypertrophic cardiomyopathy (HCM), 10 of dilated cardiomyopathy (DCM), 6 of myocarditis, and 5 of controls were studied. Right and left ventricular endomyocardial biopsy samples were obtained at the diagnostic cardiac catheterization. The samples were processed for immunohistological staining for TRX, which was done by the indirect immunoperoxidase technique. 8-hydoxy-2-deoxyguanosine (8-OHdG), one of the major DNA base-modified products, was also detected for an established marker for oxidative stress. TRX immunoreactivity was none or trivial in control specimens. Positive TRX staining was found in 6 cases; 3 in active myocarditis and 3 in DCM. The positive staining was found in infiltrating cells and damaged myocytes in the perinecrotic lesions. Damaged myocytes were also positive for 8-OHdG. All the 3 cases of DCM positive for TRX stain showed severe left ventricular hypertrophy on electrocardiogram and highly elevated left ventricular end-diastolic pressure (> 24 mmHg), suggesting the overload of oxidative stress by hemodynamic impairment. Myocardial TRX was upregulated in myocarditis and cardiomyopathies with active necrotic stage associated with DNA damage, which may reflect the oxidative stress overload in hemodynamically uncontrolled status.     

Nuclear DNA of myocardial cells in the periphery of infarctions and scars.

In 20 hearts the enlargement of muscle fibers and the increase in nuclear DNA is studied in the periphery of fresh and healed infarctions. The data of attack is verified by ECG records in most cases, five had passed undiagnosed. Selective cytophotometry is performed by measuring the largest nucleus in each of 50 fields of comparable density. Since significant differences in polyploidization exist between the control regions of hearts of similar weight primarily regions of the same heart are compared. No differences are observed in five hearts with infarctions about one week old. After five to six months the enlargement of the fibers is significaant (P less than 0.0005) while the increase in polyploidy is still slight (P less than 0.15). In all cases with scars one to nine years old the enlargement of fibers is significant (P less than 0.0005) while the increase in DNA is significant in most of them (5 X P less than 0.0005, 3 X P less than 0.005, 2 X P less than 0.01). In a case with two lesions, one posterior five (P less than 0.0005) and one anterior seven years old (P less than 0.0005), there is still slight increase in polyploidy with age (P less than 0.1). In the five undiagnosed cases only one has no significant increase in DNA. There is no correlation found between the enlargement of fibers and the increase in polyploidy in this limited number of cases.     

Mitotic polyploidization of mouse heart myocytes during the first postnatal week

Summary Polyploidization of myocytes in the cardiac ventricle of mice occurs predominantly during the first week of the postnatal life. Using isolated cells it was shown that about 70 % of the cardiomyocytes become binuclear at this time (2c×2), while 10 % are mononuclear but contain 4c of DNA, where c was the haploid level of DNA. About 2 % of the cell population were represented by 4c×2 or 8c cells.Cytophotometry of Feulgen-stained DNA in ¹⁴C-thymidine-labeled nuclei has shown that the cells that enter the mitotic cycle are mainly diploid. After mitosis (30 h or more after thymidine application) the label was found predominantly in 2c×2 and 4c cell types. Comparison of the curves presenting dynamics of labeled mitoses and the accumulation of labeled binuclear cells reveals that binuclear 2c×2 cells are formed by acytokinetic mitosis. The formation of 4c mononuclear cells is accomplished via other types of mitotic arrest; this may be due, for example, to a block in the pro-or metaphase.Only very rare cases of cytotomy were detected and the number of newly formed 2c cells was very low. It is concluded that cell multiplication is practically arrested at this period of life, and growth of the ventricular mass is due to polyploidization of virtually all cycling cells, while their number remains unchanged. Mechanisms and functional significance of cardiomyocyte polyploidization are discussed.     

Similarities and differences in the growth of the heart in situ and in transplants

Growth and ploidy of rat ventricular myocytes were studied during development in situ and in grafts (1 day old rat ventricle transplanted under kidney capsule of syngenic adult animals). Both in situ and in the transplants polyploidization occurred on days 4-14 of postnatal life, and the modal group of myocytes was represented by binucleate diploid (2c x 2) cells. Minor quantities of 4c, 4c x 2, 8c and 2c x 4 myocytes were detected as well. In ventricles of 14 and 28 days old rats and in the transplants of the corresponding age the portion of polyploid myocytes was 90-96% and 32-63% respectively. The intensity of postmitotic myocyte transplants was decreased as compared with in situ development, and cells that exit proliferation cycle did not grow until day 14. The data on thymidine label dilution suggest that diploid myocytes of the transplant can divide two or three times but the majority of labeled diploids divided only once. Labeled 2c x 2 myocytes originated from the first, and less frequently, the second cell generation or resulted from initial acytokinetic mitosis. Mononucleate tetraploids 4c originated from 2c x 2 and mostly from 2c cells. Octaploids were formed after 3d or 4th labeled mitosis. The conclusion about cardiac myocyte polyploidization as an intrinsic developmental program is supported, implying the programming of onset, mode, duration and termination of polyploidization and its prolongation during early postnatal life.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical overload-induced apoptosis: A study in cultured neonatal ventricular myocytes and fibroblasts

Apoptosis of cardiac myocytes has been implicated in cardiac dysfunction due to chronic hemodynamic overload. Reports on the role of apoptosis in the transition from hypertrophy to decompensated heart failure are not unequivocal. In this study we analysed the direct relationship between mechanical overload and induction of apoptosis in an in vitro model of cultured heart cells. Cyclic mechanical stretch was applied to cultured neonatal rat ventricular myocytes and fibroblasts. Several indicators of apoptosis were examined, such as morphological features, caspase-3 activity and DNA fragmentation. Mechanical strain did not induce any significant change in these parameters as compared to non-stretched myocytes or fibroblasts. However, administration of staurosporine, a known inducer of apoptosis, induced massive apoptosis in myocytes as well as fibroblasts. We conclude that this in vitro cell model system lacks a direct link between mechanical stretch and apoptosis. The three-dimensional structure-function relationship of myocardial tissue in the intact heart may elicit stretch-induced molecular signaling cascades in a much more complex way than in monolayer cultures of cardiac cells.     

Somatic polyploidy promotes cell function under stress and energy depletion: evidence from tissue-specific mammal transcriptome

Polyploid cells show great among-species and among-tissues diversity and relation to developmental mode, suggesting their importance in adaptive evolution and developmental programming. At the same time, excessive polyploidization is a hallmark of functional impairment, aging, growth disorders, and numerous pathologies including cancer and cardiac diseases. To shed light on this paradox and to find out how polyploidy contributes to organ functions, we review here the ploidy-associated shifts in activity of narrowly expressed (tissue specific) genes in human and mouse heart and liver, which have the reciprocal pattern of polyploidization. For this purpose, we use the modular biology approach and genome-scale cross-species comparison. It is evident from this review that heart and liver show similar traits in response to polyploidization. In both organs, polyploidy protects vitality (mainly due to the activation of sirtuin-mediated pathways), triggers the reserve adenosine-5′-triphosphate (ATP) production, and sustains tissue-specific functions by switching them to energy saving mode. In heart, the strongest effects consisted in the concerted up-regulation of contractile proteins and substitution of energy intensive proteins with energy economic ones. As a striking example, the energy intensive alpha myosin heavy chain (providing fast contraction) decreased its expression by a factor of 10, allowing a 270-fold increase of expression of beta myosin heavy chain (providing slow contraction), which has approximately threefold lower ATP-hydrolyzing activity. The liver showed the enhancement of immunity, reactive oxygen species and xenobiotic detoxication, and numerous metabolic adaptations to long-term energy depletion. Thus, somatic polyploidy may be an ingenious evolutionary instrument for fast adaptation to stress and new environments allowing trade-offs between high functional demand, stress, and energy depletion.     

Electrical Inhomogeneity in Left Ventricular Hypertrophy

Recent studies designed to assess the relationship between aortic compliance and heterogeneity of heart electrical activity has shown that hypertrophy aggravates repolarization disturbances in the myocardium. Numerous mechanisms of electrical instability and inhomogeneity associated with left ventricular hypertrophy are now under investigation. Most of the studies have been found to be focused on ventricular Gradient, QT dispersion, amplitudes of isointegral maps during ventricular repolarization, abnormally low-QRST areas, dispersion of the QT interval, and spatial QRS-T _angle. These studies point to marked repolarization abnormalities in left ventricular hypertrophy and the dispersion of the QT interval as a valuable index for inhomogeneity of repolarization and the subsequent heart rate variability. The heart rate-corrected QT dispersion and QT apex dispersion seem to be significantly longer in the patients with left ventricular hypertrophy than in normal individuals. The review study has also identified QRST isointegral map as a valuable technique in assessment of the electro-cardiac events in LVH.     

Polyploidization and endomitosis in giant cells of rabbit trophoblast

Morphological and cytophotometric investigations have been performed on giant cells of the rabbit trophoblast to reveal a mechanism of nuclei polyploidization and define the level of polyploidy. The character of endomitotic chromosomes is found to differ and depend largely on the degree of nuclei polyploidy. Small chromosomes were found in nuclei with low levels of polyploidy. For highly polyploid nuclei, two stages are distinguished. In the first case condensed chromosomes join into bundles resembling Riesenchromosomen in plants, whereas in the second, decondensed chromosomal threads separate and disperse in the karyoplasm. The splitting does not involve nuclei-forming chromosomes in the region of the nucleolar organiser. The degree of polyploidy was determined on the 15th day of development. It was found that giant cell nuclei contain DNA in amounts corresponding to 32-512 chromosomal sets. Most of the nuclei have levels of 128c and 256c. Highly-polyploid nuclei disintegrate into small nuclei with the degree of polyploidy varying from 1c to 32c. Di- tri- and tetraploid nuclei predominate.     

Urocortin, a member of the corticotropin-releasing factor family, in normal and diseased heart

In the present study we investigated the form of expression, action, second messenger, and the cellular location of urocortin, a member of the corticotropin-releasing factor (CRF) family, in the heart. Urocortin mRNA, as shown by quantitative RT-PCR analysis, is expressed in the cultured rat cardiac nonmyocytes (NMC) as well as myocytes (MC) in the heart, whereas CRF receptor type 2beta (CRF-R2beta), presumed urocortin receptor mRNA, is predominantly expressed in MC compared with NMC. Urocortin mRNA expression is higher in left ventricular (LV) hypertrophy than in normal LV, whereas CRF-R2beta mRNA expression is markedly depressed in LV hypertrophy compared with normal LV. Urocortin more potently increased the cAMP levels in both MC and NMC than did CRF, and its effect was more potent in MC than in NMC. Urocortin significantly increased protein synthesis by [(14)C]Phe incorporations and atrial natriuretic peptide secretion in MC and collagen and increased DNA synthesis by [(3)H]prolin and [(3)H]Thy incorporations in NMC. An immunohistochemical study revealed that urocortin immunoreactivity was observed in MC in the normal human heart and that it was more intense in the MC of the human failing heart than in MC of the normal heart. These results, together with the recent evidence of urocortin for positive inotropic action, suggest that increased urocortin in the diseased heart may modulate the pathophysiology of cardiac hypertrophy or failing heart, at least in part, via cAMP signaling pathway.     

A decrease in cyclin B1 levels leads to polyploidization in DNA damage‐induced senescence

Adriamycin, an anthracycline antibiotic, has been used for the treatment of various types of tumours. Adriamycin induces at least two distinct types of growth repression, such as senescence and apoptosis, in a concentration‐dependent manner. Cellular senescence is a condition in which cells are unable to proliferate further, and senescent cells frequently show polyploidy. Although abrogation of cell division is thought to correlate with polyploidization, the mechanisms underlying induction of polyploidization in senescent cells are largely unclear. We wished, therefore, to explore the role of cyclin B1 level in polyploidization of Adriamycin‐induced senescent cells. A subcytotoxic concentration of Adriamycin induced polyploid cells having the features of senescence, such as flattened and enlarged cell shape and activated β‐galactosidase activity. In DNA damage‐induced senescent cells, the levels of cyclin B1 were transiently increased and subsequently decreased. The decrease in cyclin B1 levels occurred in G2 cells during polyploidization upon treatment with a subcytotoxic concentration of Adriamycin. In contrast, neither polyploidy nor a decrease in cyclin B1 levels was induced by treatment with a cytotoxic concentration of Adriamycin. These results suggest that a decrease in cyclin B1 levels is induced by DNA damage, resulting in polyploidization in DNA damage‐induced senescence.