Cytotoxic potential of the phytochemical genistein isoflavone (4',5',7-trihydroxyisoflavone) and certain environmental chemical compounds on testicular cells

The effects of genistein (Gn), sodium azide (naz), and dexamethasone (dxm) on testicular cells TM3, TM4 and GC-1 spg were studied in vitro. First, a series of experiments were performed to assess the response of the cells to the exposure of Gn, naz, dxm, a combination of Gn with naz and Gn with dxm. Trypan blue exclusion assay was used to determine the percentage of viability, and LDH-cytotoxicity test was used to assess the degree of treatment-induced cytotoxicity on each cell type. A second series of experiments were performed to study cytomorphology and determine the type and percentage of treatment-induced cell death (apoptosis and necrosis) on each cell line, using fluorescent dye technique to detect apoptotic and necrotic cells, and tunnel assay to confirm apoptosis. The results from the data obtained demonstrated: i) that incubation of testis cells with each of the agents (Gn, dxm, naz) alone and in two combinations (Gn-dxm, and Gn-naz) induced significant testicular cell death; ii) that both genistein and dexamethasone mostly and significantly induced apoptotic cell death while sodium azide induced necrotic cell death; iii) that addition of dexamethasone to genistein demonstrated synergism in apoptosis on testis cells; and iv) that combination of naz with Gn demonstrated synergism in necrosis on testis cells even though Gn alone did not induce significant necrosis. It is concluded that the synergistic actions of genistein and dxm, and of genistein + sodium azide in induction of apoptosis and/or necrosis may be of clinical and pathophysiological research interest considering the chemopreventive and chemotherapeutic potential of genistein; and the clinico-pharmacological application of dexamethasone and sodium azide.     

Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells

The role of caspase-3 (CPP32) protease in the molecular pathways of genistein-induced cell death in TM4 cells was investigated. Fluorescence microscopy with Hoechst-33258-PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the test cells was assessed with both the trypan blue exclusion and MTT tetrazolium (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetralzolium bromide, 2.5 mg/mL) assays. Caspase-3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit. The overall results from all the data demonstrated that: i) genistein exerts dose- and time-dependent effects on TM4 testis cells; ii) apoptosis is induced by lower concentrations of genistein and necrosis induced by higher concentrations of genistein; iii) genistein induced activation caspase-3 enzymatic activity; iv) genistein-induction of apoptosis and necrosis was significantly inhibited by the caspase-3 inhibitor, z-DEV-FMK; v) sodium azide induced necrosis without activation of CPP32 enzymatic activity, and induction of apoptosis; and vi) genistein-induced apoptosis was associated with activation of CPP32 enzymatic activity in the cells. The overall results indicate a strong evidence of caspase-3 (CPP332) mediation in the molecular pathways of genistein-induced apoptosis in testicular cells. Apoptosis is the physiologically programmed cell death in which intrinsic mechanisms participate in the death of the cell, in contrast to necrosis, which induces inflammatory response in the affected cell. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis augments the biotherapeutic potential of genistein for the treatment of malignant diseases including prostate and testicular cancers. It is therefore inevitable that identification of the apoptotic pathways and the points at which regulation occurs could be instrumental in the design of genistein biotherapy for such diseases.     

Genistein Enhances the Cisplatin‐Induced Inhibition of Cell Growth and Apoptosis in Human Malignant Melanoma Cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy‐refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 μM) did not induce apoptosis by itself but did enhance cisplatin‐induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti‐apoptotic proteins, bcl‐2 and bcl‐xL, and one pro‐apoptotic protein, apoptotic protease activating factor‐1 (Apaf‐1), were examined. Genistein alone or cisplatin alone generally did not alter bcl‐2 expression or bcl‐xL expression, but slightly increased Apaf‐1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl‐2 and bcl‐xL protein and increased Apaf‐1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.     

Phenomena of Spontaneous Proliferation, Differentiation, and Apoptosis in Primary Culture of Leukemic Lymphocytes

The proliferative capacity of lymphocytes from peripheral blood of bovine with chronic lymphocytic leukemia (CLL) in vitro was investigated. We have shown earlier that CLL cells spontaneously proliferate in serum-free medium in the absence of added growth factors and mitogenic stimulation; autocrine growth factors provide the growth-initiating signal for CLL cells. The results of the present study showed that bovine serum albumin or fetal calf serum greatly enhanced the number of CLL cells incorporating [³H]thymidine. Although some CLL cells proceeded through more than one cell cycle, proliferation of CLL cells in culture was temporary. On the other hand, it was shown that CLL cells differentiated spontaneously in culture. This differentiation was characterized by the appearance of plasmacytoid cells possessing cytoplasmic immunoglobulins that coincided with the cessation of cell proliferation. Moreover, together with spontaneous proliferation and differentiation, the phenomenon of programmed cell death (apoptosis) was found, as was evidenced by the appearance of apoptotic bodies as well as DNA fragmentation. The findings indicate that the loss of proliferative potential of CLL cells in culture may be a consequence of their differentiation and/or apoptosis in vitro. CLL cells, with an autotrine growth mechanism, spontaneous differentiation, and apoptosis in vitro, provide a new model system for studies of the relationship between cellular proto-oncogene expression and inhibition of growth and/or induction of differentiation.     

Chrysophanol exhibits inhibitory activities against colorectal cancer by targeting decorin

Colorectal cancer (CRC) is a common human malignancy that accounts for 600,000 deaths annually worldwide. Chrysophanol, a naturally occurring anthraquinone compound, exhibits anti-neoplastic effects in various cancer cells. The aim of this study was to explore the biological effects of chrysophanol on CRC cells, and determine the underlying mechanism. Chrysophanol inhibited proliferation of and promoted apoptosis in CRC cells by activating the intrinsic mitochondrial apoptotic pathway. In addition, chrysophanol also suppressed tumor growth in vivo and increased the percentage of apoptotic cells in tumor xenografts, without general toxicity. Proteomic iTRAQ analysis revealed decorin (DCN) as the major target of chrysophanol. DCN was upregulated in the tumor tissues following chrysophanol treatment, and ectopic DCN expression markedly augmented the pro-apoptotic effects of chrysophanol in CRC cells. In contrast, DCN knockdown significantly abrogated chrysophanol-induced apoptosis in CRC cells. Taken together, chrysophanol exerts anti-neoplastic effects in vitro and in vivo in CRC cells by modulating DCN, there by highlighting its therapeutic potential in CRC.     

Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo

Swainsonine, an extract from Astragalus membranaceus, is known for its anti-cancer effects and could prevent metastases. In order to investigate the effects and mechanisms of swainsonine in C6 glioma cells, we carry out correlated experiments in vitro and in vivo. After treatment with swainsonine, the effective dose and IC₅₀ value of swainsonine in the C6 glioma cell were examined using the MTT assay. Cell cycle distribution and apoptotic rates were analyzed using FCM and [Ca²⁺]_i was measured by LSCM. Expressions of p16 and p53 protein were evaluated by immunocytochemical methods. Simultaneously, glioma-bearing rats were administered swainsonine at doses of 2, 4 and 8 mg/kg body wt. The inhibition rate was calculated and pathological sections were observed. The results indicated that the growth of C6 glioma cells is inhibited by swainsonine in vitro, with an IC₅₀ value within 24 h of 0.05 μg/ml. Increases in swainsonine correlate with S phase percentages of 11.3%, 11.6% and 12.4%, respectively. Moreover, the expression of apoptosis inhibiting p53 and p16 protein decreases gradually. Tumor weight in vivo decreased clearly and HE dyeing of tumor tissue showed gray, its texture was soft, with necrosis and hemorrhagic concentrated inward. Swainsonine could inhibit the proliferation of C6 glioma cells in vitro and the growth of C6 glioma in vivo. The mechanisms of swainsonine-induced apoptosis may relate with the expression of apoptosis-related genes and overloading-[Ca²⁺]_i-induced endoplasmic reticulum stress.     

The protective effect of zinc on morphine-induced testicular toxicity via p53 and Akt pathways: An in vitro and in vivo approach

BackgroundChronic use of morphine is associated with reproductive complications, such as hypogonadism and infertility. While the side effects of morphine have been extensively studied in the testis, much less is known regarding the effects of morphine on Sertoli cells and the effects of zinc on morphine-induced testicular injury as well as their underlying mechanisms. Therefore, the purpose of this study was to investigate the effect of morphine (alone and co-administered with zinc) on cell viability and apoptosis of the testicular (Sertoli) cells as well as the tumor suppressor p53 and phosphorylated-protein kinase B (p-Akt) protein levels in both in vitro and in vivo models.MethodsCultured Sertoli cells were exposed to morphine (23 μM), zinc (8 μM), and zinc prior to morphine and their effects on Sertoli cell viability and apoptosis were investigated. Morphine (3 mg/kg) and zinc (5 mg/kg, 1 h before morphine) were also injected intraperitoneally to rats and then the apoptotic changes in the testis were evaluated.ResultsCell viability and p-Akt protein levels decreased in morphine-treated cells, while apoptosis and p53 protein expression increased in these cells. Pretreatment with zinc recovered morphine-induced apoptotic effects, as well as over-expression of p53 and down-regulation of p-Akt. These findings were supported by a subsequent animal study.ConclusionThe present data indicated the protective effect of zinc against morphine-induced testicular (Sertoli) cell toxicity via p53/Akt pathways in both in vivo and in vitro models and suggested the clinical importance of zinc on infertility among chronic opioid users and addicted men.     

iPSC‐derived mesenchymal stem cells exert SCF‐dependent recovery of cigarette smoke‐induced apoptosis/proliferation imbalance in airway cells

Mesenchymal stem cells (MSCs) have emerged as a potential cell‐based therapy for pulmonary emphysema in animal models. Our previous study demonstrated that human induced pluripotent stem cell–derived MSCs (iPSC‐MSCs) were superior over bone marrow–derived MSCs (BM‐MSCs) in attenuating cigarette smoke (CS)‐induced airspace enlargement possibly through mitochondrial transfer. This study further investigated the effects of iPSC‐MSCs on inflammation, apoptosis, and proliferation in a CS‐exposed rat model and examined the effects of the secreted paracrine factor from MSCs as another possible mechanism in an in vitro model of bronchial epithelial cells. Rats were exposed to 4% CS for 1 hr daily for 56 days. At days 29 and 43, human iPSC‐MSCs or BM‐MSCs were administered intravenously. We observed significant attenuation of CS‐induced elevation of circulating 8‐isoprostane and cytokine‐induced neutrophil chemoattractant‐1 after iPSC‐MSC treatment. In line, a superior capacity of iPSC‐MSCs was also observed in ameliorating CS‐induced infiltration of macrophages and neutrophils and apoptosis/proliferation imbalance in lung sections over BM‐MSCs. In support, the conditioned medium (CdM) from iPSC‐MSCs ameliorated CS medium‐induced apoptosis/proliferation imbalance of bronchial epithelial cells in vitro. Conditioned medium from iPSC‐MSCs contained higher level of stem cell factor (SCF) than that from BM‐MSCs. Deprivation of SCF from iPSC‐MSC‐derived CdM led to a reduction in anti‐apoptotic and pro‐proliferative capacity. Taken together, our data suggest that iPSC‐MSCs may possess anti‐apoptotic/pro‐proliferative capacity in the in vivo and in vitro models of CS‐induced airway cell injury partly through paracrine secretion of SCF.     

Paclitaxel administration and its effects on clinically relevant human cancer and non cancer cell lines

Comparisons of the effects of clinically relevant concentrations of the anticancer agent paclitaxel on growth, viability, and apoptosis were determined using in vitro human cell cultures. Growth of the cervical cancer cell line, HeLa-S3, was significantly reduced, and apoptotic index was significantly increased, after 24 h in cultures treated with 12 nM paclitaxel. In contrast, hepatic carcinoma (HEpG2) cells capable of detoxifying paclitaxel were only affected at paclitaxel concentrations ge120 nM. The previously uncharacterized non-cancerous human microvessel endothelial cell line HMEC-1, was more sensitive to paclitaxel treatment than both HeLa-S3 and HEpG2 cells, demonstrating decreased growth and increased apoptosis with 1.2 nM paclitaxel. These results are significant in the design of in vitro cell culture systems to study drug metabolism and toxicity.     

Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle

Apoptosis and cell proliferation are accepted to be responsible for the maintenance of homeostasis in the hematopoietic system. Understanding of the mechanisms of action of the aminothiols and ionizing radiation on normal hematopoietic cells requires determination of the correlation between apoptotic cell death and cell cycle distribution. The effects of WR-2721 ((S)-2-/3-aminopropylamino/ethylphosphorothioic acid; Amifostine) and ⁶⁰Co gamma-rays on apoptosis and cell cycle progression in the mouse bone marrow were determined. Adult male Swiss mice were exposed to 6 Gy gamma-rays only, or pretreated with WR-2721, at a dose of 400 mg/kg body weight, 30 min before gamma-irradiation. The laser scanning cytometry APO-BRDU^TM assay based on simultaneous analysis of cellular DNA content and the in situ detection of DNA strand breaks was used to identify apoptotic cells and to reveal the cell cycle position of apoptotic and nonapoptotic cells. Temporary changes in the frequency of apoptotic cells with fluorescein isothiocyanate (FITC) labeling of DNA strand breaks, and all bone marrow cells including apoptotic and nonapoptotic ones, whose DNA stained with propidium iodide, were observed in the particular phases of the cell cycle throughout the 96-h period after WR-2721 application and gamma-irradiation. The cell cycle phase specificity of WR-2721 and ⁶⁰Co gamma-irradiation was shown in terms of induction of apoptosis in bone marrow cells. The patterns of alterations in the frequency of apoptotic cells and all bone marrow cells with respect to their cell cycle position were dependent on the agent(s) applied and the time interval after treatment of mice with WR-2721 and/or gamma-rays. A modulatory, suppressive action of WR-2721 on apoptosis induction and the cell cycle perturbation caused in normal cells of the mouse bone marrow by gamma-rays was found.     

Ghrelin inhibits proliferation and increases T-type Ca channel expression in PC-3 human prostate carcinoma cells

Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca²⁺ levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca²⁺ channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca²⁺ channel expression.     

Induction of apoptosis and inhibition of cell adhesive and invasive effects by tanshinone IIA in acute promyelocytic leukemia cells in vitro

Tanshinone IIA, a diterpene quinone extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is used widely and successfully in clinics in China for treating inflammatory diseases. Recently tanshinone IIA has been reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, the anti-proliferation and apoptosis inducing effects of tanshinone IIA as well as its influence on cell adhesion to and invasion through the extracellular matrix (ECM) on acute promyelocytic leukemia (APL) NB4 cells in vitro were studied. Cell proliferation was assessed by MTT assay, cell apoptosis was observed by Hoechst 33258 staining and flow cytometry (FCM); The variation of caspase-3 and apoptotic related genes were assayed by Western blotting, cell mitochondrial membrane potential as well as cell adhesive and invasive effects were also investigated by using standard methods. The results showed that tanshinone IIA exhibited induction of apoptosis by activation of caspase-3, downregulation of anti-apoptotic protein bcl-2 and bcl-xl and upregulation of pro-apoptotic protein bax, as well as disruption of the mitochondrial membrane potential. Furthermore, treatment by tanshinone IIA could reduce cell adhesion to and invasion through ECM in leukemia NB4 cells. These data provide a potential mechanism for tanshinone IIA-induced apoptosis and cell growth inhibition in leukemia NB4 cells, suggesting that tanshinone IIA may serve as an effective adjunctive reagent for the treatment of APL.Contributed equally to this study.     

Dual effects of isoflavonoids from Pueraria lobata roots on estrogenic activity and anti-proliferation of MCF-7 human breast carcinoma cells

Pueraria lobata root (PLR), well known as Kudzu root, has recently become commercially available in Western dietary supplements for menopausal symptoms. The scientific basis for its action has been attributed to the action of phytoestrogens. This study aimed to investigate the estrogen-like activity of isoflavonoids isolated from P. lobata root and their safety with respect to their effect on breast cancer cell proliferation. In an E-screen assay, crude MeOH extract of PLR significantly increased the proliferation of MCF-7 cells in a concentration-dependent manner. Among the four fractions obtained by solvent fractionation of MeOH extract, the n-BuOH fraction had significant estrogen-like activities at all concentrations tested. Phytochemical analysis of the n-BuOH fraction led to the isolation of 10 isoflavones (1–10), among which genistein (10) had significant estrogen-like activities at all concentrations tested. These activities were significantly enhanced by treatment with genistein and 17β-estradiol compared with 17β-estradiol alone, and this effect was mediated by decreased expression of estrogen receptor (ER)α and phospho-ERα in MCF-7 cells. In a cell cytotoxicity assay, genistein (10) exhibited significant cytotoxicity in both ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. This cytotoxicity was characterized by the induction of apoptotic cells stained with annexin V conjugated with Alexa Fluor 488 and involved activation of mitochondria-independent and -dependent apoptosis pathways in MCF-7 cells. Our results demonstrated that genistein (10) has estrogen-like effects dependent on ER pathway activation and anti-proliferative effects mediated by the apoptosis pathway rather than the ER pathway in MCF-7 breast cancer cells.     

Controversial effect on Erk activation of some cytotoxic drugs in human LOVO colon cancer cells

The use of some classic antibiotics was recently shown to inhibit growth and to induce apoptosis in human LOVO colon cancer cells. In this study, we describe that ciprofloxacin (CI), trimebutine maleate (COL) and tiemonium methylsulfate (VIS) greatly inhibit cell proliferation in vitro. Proliferation inhibition reached its maximum at 10^–4?M, 10^–3?M and 10^–2?M, respectively, for COL, CI and VIS. Moreover, phospho-extracellular-regulated kinase was totally abrogated in non-apoptotic cytotoxicity of VIS but decreases or increases in the apoptotic inhibition, respectively, of COL and CI treatments.

Abbreviations: CI: ciprofloxacin; COL: trimebutine maleate; VIS: tiemonium methylsulfate; MAPK/Erk: mitogen-activated protein kinases/extracellular-regulated kinase     

Functional role of the Ca-activated Cl channel DOG1/TMEM16A in gastrointestinal stromal tumor cells

DOG1, a Ca²⁺-activated Cl⁻ channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl⁻ currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.     

Genistein对大鼠垂体前叶细胞增殖的抑制作用

应用细胞培养、^3H-TdR掺入、流式细胞和电镜技术,观察酪氨酸蛋白激酶（PTK）抑制剂genistein对正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20增殖的影响,并探讨其可能的机制。结果显示：genistein作用48h后可明显抑制正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20增殖。流式细胞仪检测发现,50和100μmol/L genistein可将AtT-20细胞阻断于G0／G1期及G2／M期,并出现凋亡峰,凋亡率分别灰19.9％和36.4％。电镜照片显示有凋亡细胞。结果表明,PTK抑制剂可以明显抑制正常大鼠垂体前叶细胞和垂体瘤细胞株AtT-20的殖,并诱导细胞凋亡,说明PTK活性对细胞增殖和分化有重要作用。     

蛋氨酸脑啡肽抑制人胃癌细胞BGC823增殖作用及免疫调节机制

采用不同浓度梯度的蛋氨酸脑啡肽(methionine enkephalin,MENK)体外作用于人胃癌细胞BGC823后,探讨对其增殖影响及其作用机制,为胃癌的免疫治疗提供理论依据。体外培养人胃癌细胞株BGC823,PCR检测阿片受体OGFr的表达;用不同浓度(0、1、2、3、4 mg/mL)的MENK体外作用于BGC823细胞24、48、72、96 h后,MTS检测MENK对其增殖影响;流式细胞术和Annexin V-FITC/PI双染法检测4 mg/mL MENK体外处理48、72 h后BGC823细胞凋亡变化。结果显示,人胃癌BGC823细胞有阿片受体OGFr的表达;MENK可抑制BGC823细胞增殖,且随着剂量的增加和时间的延长,其抑制作用逐渐增强(P0.05);4 mg/mL MENK48 h处理组与空白组相比细胞凋亡率增加,72 h处理组与48 h处理组结果一致(P0.05)。结果表明,MENK可抑制BGC823细胞增殖,具有显著的剂量依赖性和时间依赖性,且可通过诱导细胞凋亡抑制BGC823细胞的增殖。     

3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein

[³H]-thymidine is commonly used to analyze the accumulation of [³H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [³H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [³H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [³H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [³H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [³H]-thymidine-labeled DNA. They also demonstrate that [³H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na⁺] _i /[K⁺] _i ratio.