Regulation Of Cell Cycle-related Proteins Research Articles

Tripartite motif containing 62 is a novel prognostic marker and suppresses tumor metastasis via c-Jun/Slug signaling-mediated epithelial-mesenchymal transition in cervical cancer.

BackgroundTRIM62 (tripartite motif containing 62) has been found to act as a tumor suppressor of several cancers. However, its precise biological role and related mechanism remain unknown in cervical cancer (CC).MethodsQuantitative Real-time PCR and western blot were adopted to detect the mRNA and protein expression level of TRIM62 in both human CC cell lines and tissues. Immunohistochemistry was used to measure the TRIM62 expression in 30 normal cervical and 189 CC tissues. Univariate and multivariate Cox regression analyses and Kaplan–Meier survival analyses performed to investigate the association between TRIM62 expression and CC patients’ prognosis. The effect of TRIM62 on CC growth and metastasis was studied in vitro and in vivo. Multi-pathway reporter array were utilized to identify the potential signaling manipulated by TRIM62.ResultsTRIM62 was frequently down-regulated in both human CC cells and tissues. Low expression of TRIM62 in CC tissues was associated with aggressive clinicopathological features of CC patients. In addition, TRIM62 was also an independent poor prognostic factor for overall and disease-free survival of CC patients after surgery. Moreover, enforced expression of TRIM62 in CC cells significantly inhibited their abilities of proliferation, migration and invasion in vitro. Besides, subcutaneous xenograft tumor model and xenograft mouse metastatic model respectively displayed that TRIM62 impeded the growth and metastasis of CC in vivo. Furthermore, mechanism study exhibited that TRIM62 could suppress epithelial-mesenchymal transition (EMT) by inhibiting c-Jun/Slug signaling. The inhibitory role of TRIM62 in tumor proliferation might be through regulating cell cycle related proteins CyclinD1 and P27 by targeting c-Jun.ConclusionTRIM62 is a potential prognostic biomarker in CC and suppresses metastasis of CC via inhibiting c-Jun/Slug signaling-mediated EMT.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0445-5) contains supplementary material, which is available to authorized users.

Coptis japonicaMakino extract suppresses angiogenesis through regulation of cell cycle-related proteins

Angiogenesis, neovascularization from pre-existing vessels, is a key step in tumor growth and metastasis, and anti-angiogenic agents that can interfere with these essential steps of cancer development are a promising strategy for human cancer treatment. In this study, we characterized the anti-angiogenic effects of Coptis japonica Makino extract (CJME) and its mechanism of action. CJME significantly inhibited the proliferation, migration, and invasion of vascular endothelial growth factor (VEGF)-stimulated HUVECs. Furthermore, CJME suppressed VEGF-induced tube formation in vitro and VEGF-induced microvessel sprouting ex vivo. According to our study, CJME blocked VEGF-induced cell cycle transition in G1. CJME decreased expression of cell cycle-regulated proteins, including Cyclin D, Cyclin E, Cdk2, and Cdk4 in response to VEGF. Taken together, the results of our study indicate that CJME suppresses VEGF-induced angiogenic events such as proliferation, migration, and tube formation via cell cycle arrest in G1.

AB109. Downregulation of tNASP inhibits proliferation through regulating cell cycle-related proteins and inactive ERK/MAPK signal pathway in renal cell carcinoma cells

ObjectiveNuclear auto-antigenic sperm protein (NASP), initially described as a highly auto-immunogenic testis and sperm-specific protein, is a histone chaperone that is proved to present in all dividing cells. NASP has two splice variants: testicular NASP (tNASP) and somatic form of NASP (sNASP). Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP. Up to now, little has been known about tNASP in renal cell carcinoma (RCC). In the present study, the molecular mechanism of tNASP in RCC was explored.MethodsThe expression level of tNASP in 16 paired human RCC specimens was determined. Downregulation of tNASP by small interfering RNA (siRNA) was transfected in RCC cell lines. The effect of downregulation of tNASP by siRNA on cell colony formation and proliferation was examined by colony formation assay and CCK-8 assay, cell cycle was analyzed by flow cytometry, and the expression of cyclin D1 and P21 were detected by Western blotting. ERK/MAPK signaling was also analyzed.ResultstNASP has a relative high expression level in human RCC tissues. Via upregulation of P21 and downregulation of cyclinD1, silence of tNASP can inhibit cell proliferation, which induces cell cycle arrest. Furthermore, ERK signaling pathway is confirmed to mediate the regulation of cell cycle-related proteins caused by silence of tNASP.ConclusionsOur research demonstrates that knockdown of tNASP effectively inhibits the proliferation and causes G1 phase arrest through ERK/MAPK signal pathway.

Abstract 5385: Anticancer effects of a novel histone deacetylase inhibitor, SNUCH-1, in glioblastoma

Abstract Histone deacetylase inhibitors (HDACi) have been evaluated as promising anticancer drugs against glioblastoma cells. However, there are still lack of animal experiments and new HDAC inhibitors for clinical trials. In this study, we demonstrated the therapeutic efficacy of novel pan-HDACi, SNUCH-1, comparing with other traditional pan-HDACi such as trichostatin A (TSA) and vorinostat (SAHA) in vitro and vivo. We performed cell viability, proliferation, cell cycle, apoptosis, HDAC enzyme activity and western blot assay using the three glioblastoma cell lines and two primary cultured cells. Furthermore, we examined the effects of SNUCH-1 by intracisternal administration on the orthotopic human xenograft glioblastoma mice model. We showed that SNUCH-1 decreased cell viability in a dose-dependent manner and suppressed proliferation with a stronger apoptotic activity than TSA and SAHA. SNUCH-1 inhibited HDAC enzyme activity and blocked cell cycle progression at G2/M, regulating cell cycle related protein expression more effectively than TSA and SAHA. In addition, SNUCH-1 significantly reduced tumor volume and revealed more powerful anti-tumor effect in vivo than TSA. Our results suggest that the novel HDAC inhibitor, SNUCH-1, is a potent therapeutic candidate for glioblastoma. Key words: Histone deacetylase inhibitor, glioblastoma, intracisternal administration Citation Format: Seung Ah Choi, Phi Ae Kwak, Young Eun Lee, Ji Hoon Phi, Jung Won Choi, Kyu-Chang Wang, Chul-Kee Park, Seung-Ki Kim. Anticancer effects of a novel histone deacetylase inhibitor, SNUCH-1, in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5385. doi:10.1158/1538-7445.AM2015-5385

Downregulation of tNASP inhibits proliferation through regulating cell cycle-related proteins and inactive ERK/MAPK signal pathway in renal cell carcinoma cells.

Nuclear auto-antigenic sperm protein (NASP), initially described as a highly auto-immunogenic testis and sperm-specific protein, is a histone chaperone that is proved to present in all dividing cells. NASP has two splice variants: testicular NASP (tNASP) and somatic form of NASP (sNASP). Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP. Up to now, little has been known about tNASP in renal cell carcinoma (RCC). In the present study, the molecular mechanism of tNASP in RCC was explored. The expression level of tNASP in 16 paired human RCC specimens was determined. Downregulation of tNASP by small interfering RNA (siRNA) was transfected in RCC cell lines. The effect of downregulation of tNASP by siRNA on cell colony formation and proliferation was examined by colony formation assay and CCK-8 assay, cell cycle was analyzed by flow cytometry, and the expression of cyclin D1 and P21 were detected by Western blotting. ERK/MAPK signaling was also analyzed. tNASP has a relative high expression level in human RCC tissues. Via upregulation of P21 and downregulation of cyclinD1, silence of tNASP can inhibit cell proliferation, which induces cell cycle arrest. Furthermore, ERK signaling pathway is confirmed to mediate the regulation of cell cycle-related proteins caused by silence of tNASP. Our research demonstrates that knockdown of tNASP effectively inhibits the proliferation and causes G1 phase arrest through ERK/MAPK signal pathway.

Anti-breast cancer activity of Fine Black ginseng (Panax ginseng Meyer) and ginsenoside Rg5

BackgroundBlack ginseng (Ginseng Radix nigra, BG) refers to the ginseng steamed for nine times and fine roots (hairy roots) of that is called fine black ginseng (FBG). It is known that the content of saponin of FBG is higher than that of BG. Therefore, in this study, we examined antitumor effects against MCF-7 breast cancer cells to target the FBG extract and its main component, ginsenoside Rg5 (Rg5). MethodsAction mechanism was determined by MTT assay, cell cycle assay and western blot analysis. ResultsThe results from MTT assay showed that MCF-7 cell proliferation was inhibited by Rg5 treatment for 24, 48 and 72 h in a dose-dependent manner. Rg5 at different concentrations (0, 25, 50 and 100 μM), induced cell cycle arrest in G0/G1 phase through regulation of cell cycle-related proteins in MCF-7 cells. As shown in the results from western blot analysis, Rg5 increased expression of p53, p21WAF1/CIP1 and p15INK4B and decreased expression of Cyclin D1, Cyclin E2 and CDK4. Expression of apoptosis–related proteins including Bax, PARP and Cytochrome c was also regulated by Rg5. These results indicate that Rg5 stimulated cell apoptosis and cell cycle arrest at G0/G1 phase via regulation of cell cycle-associated proteins in MCF-7 cells. ConclusionRg5 promotes breast cancer cell apoptosis in a multi-path manner with higher potency compared to 20(S)-ginsenoside Rg3 (Rg3) in MCF-7 (HER2−/ER+) and MDA-MB-453 (HER2+/ER−) human breast cancer cell lines, and this suggests that Rg5 might be an effective natural new material in improving breast cancer.

The overexpression of MCPH1 inhibits cell growth through regulating cell cycle-related proteins and activating cytochrome c-caspase 3 signaling in cervical cancer

MCPH1, initially identified as an hTERT repressor, has recently been implicated in mediating DNA damage response and maintaining chromosome integrity. This study is to investigate its potential role in the onset of cervical cancer. In the study, decreased expression of MCPH1 was observed in 19 of 31 cases (61.3%) at mRNA level and 44 of 63 cases (69.8%) at protein level of cervical tumor tissues compared with the paired nontumor tissues. Reduced MCPH1 protein expression was significantly associated with high-tumor grade (1 vs. 3 P = 0.013; 2 vs. 3 P = 0.047). In addition to inhibit SiHa cell migration and invasion, the overexpression of MCPH1 inhibited cervical cancer cells growth through inducing S phase arrest and mitochondrial apoptosis. Further analysis demonstrated cyclinA2/CDK2, CDC25C-cyclinB/CDC2, and p53/p21 pathways were involved in the MCPH1 overexpression-induced S phase arrest. Moreover, the overexpression of MCPH1 activated mitochondrial apoptosis through regulating several apoptosis-related proteins such as p53, Bcl-2, Bax, cytochrome c, caspase-3, and PARP-1. Our findings indicate that downregulated MCPH1 correlates with tumor progression in cervical cancer, and MCPH1 has an important role in regulating cell growth through regulating the cell cycle and apoptosis. Thus, it may be a crucial tumor suppressor gene and a novel candidate therapeutic target for cervical cancer.

Diethylstilbestrol induces arrest of rat vascular smooth muscle cell cycle progression through downregulation of cyclin D1 and cyclin E

Cardiovascular disease is associated with a multitude of pathophysiologic conditions, including vascular smooth muscle cell (VSMC) proliferation in response to vessel injury. Diethylstilbestrol (DES) was previously prescribed for at-risk pregnancies to prevent abortion, miscarriage, and premature labor. Our aim in this study was to elucidate the effects and molecular mechanism of DES on proliferation and cell cycle progression of platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. Treating the cells with DES (1-7 μM) dramatically inhibited cell proliferation in a dose-dependent manner without any cytotoxic effects. In addition, DES blocked cell cycle progression from PDGF-BB-stimulated cells, which we found was related to down-regulation of the cell cycle regulatory factors, cyclin D1, and cyclin E. Our data demonstrate that DES inhibits rat aortic VSMC proliferation and cell cycle progression through regulation of cell cycle-related proteins. Therefore, our observations may explain, in part, the mechanistic basis underlying the therapeutic effects of DES in cardiovascular disease.

Nitric oxide inhibits gastric cancer cell growth through the modulation of the Akt pathway

Nitric oxide (NO) is involved in a number of physiological and pathological processes. As an important biological mediator, NO has been the focus of cancer study for its function in tumorigenesis, tumor progression and death. The effects of NO on tumor cells are multifaceted, but many details underlying these effects are not yet well understood. In this study, we demonstrate that NO directly suppresses the growth of BGC-823 cells by inducing G0/G1 phase arrest in a dose- and time-dependent manner. We also reveal that G0/G1 arrest results from the NO-induced disruption of the cell cycle balance, which is mediated by the up-regulation of p21waf1/cip1 and the down-regulation of cyclin D1 and E and PCNA. Exposure of BGC-823 cells to various sodium nitroprusside (SNP) concentrations for 24h or to 1mmol/l SNP for various times resulted in a marked decrease in the level of phospho-Akt and increase in the level of phospho-ERK. In brief, the NO-induced cell growth suppression and G0/G1 arrest are mediated through the regulation of cell cycle-related proteins, which may depend on the inactivation of Akt signaling. This may be one mechanism through which NO inhibits gastric cancer cell growth.

Differential effect of quercetin and quercetin‐3‐glucoside on the proliferation of cancer cells

While most of flavonoids in plant and food sources exist as glycosidic‐conjugated forms, biological functions of glycones have been less studied than their aglycones. We investigated the differential anti‐proliferative effects of quercetin and its glycone, quercetin‐3‐glucoside (Q3G) on cancer cells. Q3G increased the anti‐proliferative effect of quercetin noticeably in the 7 cancer cell lines. Furthermore, treatment with Q3G induced higher degree of G1 cell cycle arrest and sub‐G1 phase of colon cancer cells than quercetin. Q3G also showed more potent activity than quercetin in decreasing the protein levels of cyclin D1, cyclin D3, CDK4, and CDK6, and increasing the mRNA and protein levels of p21CIP1in a p53‐independent manner. These results represented the increased anti‐proliferative effect of glucosylated quercetin through the regulation of cell‐cycle related proteins in G1 arrest. This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Science and Technology (MG08‐0303‐4‐0), Republic of Korea.

Corydalis yanhusuo W.T. Wang Extract Inhibits MCF-7 Cell Proliferation by Inducing Cell Cycle G2/M Arrest

Corydalis yanhusuo W.T. Wang (YHS) is a traditional Chinese herb widely prescribed for promoting blood circulation, reinforcing vital energy and alleviating pain. Our previous studies showed that an ethanol extract of YHS inhibits metastasis of breast cancer cells in vitro. In the present study, the anti-proliferative effect of the extract was determined by MTT assay and the LDH release was measured with a commercial kit. Intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨm) were monitored by CM- H(2)DCF-DA and JC-1 staining, respectively. Cell cycle was analyzed with propidium iodide (PI) staining by flow cytometry and protein expressions were measured by Western blotting. The YHS extract significantly inhibited MCF-7 cell proliferation in a dose-dependent manner. Significant increase of ROS formation and decrease of ΔΨm were observed. Furthermore, it induced MCF-7 cell cycle arrest at the G2/M phases. In addition, the p-cdc-2/cdc-2 protein expression ratio was increased while Rb and p21 protein expressions were decreased. The YHS extract inhibited MCF-7 proliferation by inducing G2/M cell cycle arrest, which might be mediated by inducing ROS formation, decreasing ΔΨm and regulating cell cycle related protein expressions.

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line. Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide (PI), mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. CIE inhibited proliferation of MHCC97H cells in a time- and dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial cells. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.

Antimycin A as a mitochondria damage agent induces an S phase arrest of the cell cycle in HeLa cells

Antimycin A (AMA), an electron transport chain inhibitor in mitochondria can produce reactive oxygen species (ROS) in cells. It has been reported that ROS may have roles in cell cycle progression via regulating cell cycle-related proteins. In the present study, we investigated the changes of the cell cycle distribution in AMA-treated HeLa cells in relation to cell cycle-related proteins. DNA flow cytometric analysis indicated that treatment with AMA significantly induced an S phase arrest of the cell cycle at 72 h. AMA decreased the expression of cyclin-dependent kinase inhibitor (CDKI), p21 and p27, CDK4, and cdc2 proteins. The expression of CDK6, cyclin D1, cyclin E, cyclin A, and cyclin B proteins was increased by 0.5 μM AMA, but was decreased by 2 and 10 μM AMA. The phosphorylation of Rb on the Ser (780) residue was increased by 0.5 μM AMA. Furthermore, treatment with AMA caused the accumulation of cells expressing cyclin A, B, and D1 proteins at the S phase of the cell cycle. However, treatment with 100 μM AMA nonspecifically extended all phases of the cell cycle. In conclusion, treatment with AMA (2, 10 and 50 μM) induced an S phase arrest of the cell cycle. An S phase arrest was accompanied by the alteration of other cell cycle-regulated proteins as well as S phase-related proteins.

Effects of Excretory/Secretory Products from Clonorchis sinensis and the Carcinogen Dimethylnitrosamine on the Proliferation and Cell Cycle Modulation of Human Epithelial HEK293T Cells

Clonorchis sinensis is one of the most prevalent parasitic helminths in Korea. Although cholangiocarcinoma can be induced by C. sinensis infection, the underlying mechanism is not clearly understood. To assess the role of C. sinensis infection in carcinogenesis, an in vitro system was established using the human epithelial cell line HEK293T. In cells exposed to the excretory/secretory products (ESP) of C. sinensis and the carcinogen dimethylnitrosamine (DMN), cellular proliferation and the proportion of cells in the G2/M phase increased. Moreover, the expression of the cell cycle proteins E2F1, p-pRb, and cyclin B was dramatically increased when ESP and DMN were added together. Similarly, the transcription factor E2F1 showed its highest level of activity when ESP and DMN were added simultaneously. These findings indicate that DMN and ESP synergistically affect the regulation of cell cycle-related proteins. Our results suggest that exposure to C. sinensis and a small amount of a carcinogen such as DMN can promote carcinogenesis in the bile duct epithelium via uncontrolled cellular proliferation and the upregulation of cell cycle-related proteins.

Epothilone B Inhibits Neointimal Formation after Rat Carotid Injury through the Regulation of Cell Cycle-Related Proteins

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial walls is an important pathogenetic factor of vascular disorders such as atherosclerosis and restenosis after angioplasty. Epothilone B, a novel potential antitumor compound, has a potent effect on preventing postangioplasty restenosis. Therefore, we established an in vivo rat carotid injury model and examined the potential effects of epothilone B on cardiovascular disease. We found that epothilone B potently prevented neointimal formation and in vivo VSMCs proliferation. In addition, we also showed that epothilone B significantly inhibited 5% fetal bovine serum (FBS)- and 50 ng/ml platelet-derived growth factor (PDGF)-BB-induced proliferation and cell cycle progression in rat aortic VSMCs. Furthermore, FBS and PDGF-BB induced the activations of extracellular signal-regulated kinases 1 and 2, Akt, phospholipase C gamma 1, and PDGF-receptor beta chain tyrosine kinase were not changed by epothilone B. However, epothilone B treatment caused a significant decrease in the level of cyclin-dependent protein kinase (CDK) 2, whereas it caused no change in the levels of cyclin E and down-regulated the phosphorylation of retinoblastoma, which plays a critical role in cell cycle regulation. Furthermore, levels of p27, an inhibitor of cyclin E/CDK2 complex, were significantly increased in VSMCs treated with epothilone B, indicating that this might be a major molecular mechanism for the inhibitory effects of epothilone B on the proliferation and cell cycle of VSMCs. These findings suggest that epothilone B can inhibit neointimal formation via the cell cycle arrest by the regulation of the cell cycle-related proteins in VSMCs.

Anticancer effects of (−)-epigallocatechin-3-gallate on ovarian carcinoma cell lines

Purpose: A constituent of green tea, (−)-epigallocatechin-3-gallate (EGCG), has been known to possess anti-cancer properties. In this study, we investigated the time-course anticancer effects of EGCG on human ovarian cancer cells to provide insights into the molecular-level understanding of growth suppression mechanism involved in EGCG-mediated apoptosis and cell cycle arrest. Methods: Three human ovarian cancer cell lines (p53 negative, SKOV-3 cells; mutant type p53, OVCAR-3 cells; and wild type p53, PA-1 cells) were used. The effect of EGCG treatment was studied via cell count assay, cell cycle analysis, FACS, Western blot, and macroarray assay. Results: EGCG exerts a significant role in suppressing ovarian cancer cell growth. Also, EGCG showed growth inhibitory effects in each cell line in a dose-dependent fashion and induced apoptosis and cell cycle arrest. The cell cycle was arrested at the G 1 phase by EGCG in SKOV-3 and OVCAR-3 cells. In contrast, the cell cycle was arrested in the G 1/S phase arrest in PA-1 cells. EGCG differentially regulated the expression of genes and proteins (Bax, p21, Retinoblastoma, cyclin D1, CDK4, Bcl-X L) more than 2-fold, showing a possible gene regulatory role of EGCG. The continual expression in p21WAF1 suggests that EGCG acts in the same way with p53 proteins to facilitate apoptosis after EGCG treatment. And Bax, PCNA, and Bcl-X are important in EGCG-mediated apoptosis. In contrast, CDK4 and Rb are not important in ovarian cancer cell growth inhibition. Conclusion: EGCG can inhibit ovarian cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of cell cycle-related proteins. Thereby, the EGCG-mediated apoptosis can be applied to an advanced strategy in the development of a potential drug against ovarian cancer.

Differential regulation of cell cycle-related proteins by CD95 engagement in thymocytes and T cell leukemic cell line, Jurkat.

CD95 engagement results in apoptosis in thymocytes and in the Jurkat human leukemic T cell line. Biochemical analyses in CD95-engaged thymocytes and Jurkat cells revealed dysregulation of the G1/S cell cycle control point. Cyclin E was upregulated upon CD95 engagement, suggesting G1-to-S progression, but there was no upregulation of cyclin A. Instead, cyclin E was degraded by caspases. In addition, c-myc that normally acts on S-phase progression through the activation of cdc25A appeared to be involved in the inhibition of S-phase progression after CD95 ligation. This implies that G1-->S progression and apoptosis are intimately linked in cells undergoing CD95 ligation. Furthermore, our data suggest that CD95-induced apoptosis occurs at the G1/S phase transition. We therefore suggest that CD95 engagement not only triggers death signals but also affects the G1/S checkpoint.

Inhibition of in vivo rat liver regeneration by 2-acetylaminofluorene affects the regulation of cell cycle-related proteins.

The effects of dietary 2-acetylaminofluorene (2-AAF) on cell cycle-related proteins was studied in regenerating livers from male Wistar rats. The levels of cyclins, cyclin dependent kinases (cdks), and related proteins were studied at different times during the first cell cycle after partial hepatectomy (PH). The frequency of proliferation cell nuclear antigen (PCNA)-positive nuclei, a marker of S phase progression, was almost zero during the first 27 hours after PH in the mitoinhibited 2-AAF-treated rats, while about 50% of the nuclei were labeled 24 hours after PH in control animals. Accordingly, Western blot tests showed markedly elevated PCNA protein levels from 18 hours to the end of S phase in untreated animals but no upregulation in response to 2-AAF. Compared with control animals, animals treated with 2-AAF showed increased levels of cdk 4 and cyclin D3 from 12 and 15 hours after PH, respectively, and altered cyclicity in cyclin D3 expression. No effects on cyclin E were observed, while the increase in cdk 2 levels in control animals during late G1/S (15-27 hours) was abolished by 2-AAF. p53 was induced by 2-AAF treatment during the same period, with a peak at 24 hours. The protein detected with p21 antibodies was highly expressed in unstimulated hepatocytes in control animals, and further increased by 2-AAF. The expression was sustained until 15 hours after PH in control rats while 2-AAF-treated animals lacked detectable protein during this period; however, a transient increase was observed at 21 hours. Thus, 2-AAF affects several parameters of cell cycle regulation of possible relevance for its inhibitory effects on hepatocyte proliferation in vivo.