Induction of apoptosis and expression of apoptosis related genes in human epithelial carcinoma cells by Helicobacter pylori VacA toxin.

Virulence factors produced by Helicobacter pylori have been known to be associated with serious gastroduodenal diseases. The aims of this study were to clarify the apoptosis-inducing properties of vacuolating cytotoxin (VacA) and examine the expression of apoptosis related proteins in human epithelial carcinoma cells expressing (AGS) or lacking (Kato III) p53. The midregion VacA homolog from H. pylori strain Q35 (Korean isolate) was cloned, expressed and sequenced. Recombinant VacA (VacA(418-799)) inhibited cell growth and induced apoptosis in gastric epithelial cells. Treatment with VacA(418-799) resulted in morphological changes and DNA fragmentation. Cell cycle analysis revealed subdiploid cells suggesting apoptosis, which was confirmed by the activation of caspase-3 and cleavage of PARP. VacA(418-799) also mediated a prolongation of the cell cycle progression in G1 phase. Furthermore, VacA(418-799) increased the expression of p53, p21(waf1/cip1) and Bax in AGS cells, but not in Kato III cells and did not affect the phosphorylation of Rb in both cell lines. These results indicate that recombinant VacA of H. pylori induces apoptosis in both Kato III and AGS cells, regardless of p53 status and suggest that VacA(418-799) mediate the development of gastric diseases through cell cycle arrest in the G1 phase. VacA(418-799) induction of apoptosis is associated with up-regulation of p53, p21(waf1/cip1), Bax in AGS cells and activation of caspase-3 in both cell lines.     

Platinum-(IV)-derivative satraplatin induced G2/M cell cycle perturbation via p53-p21(waf1/cip1)-independent pathway in human colorectal cancer cells

Aim:

Platinum-(IV)-derivative satraplatin represents a new generation of orally available anti-cancer drugs that are under development for the treatment of several cancers. Understanding the mechanisms of cell cycle modulation and apoptosis is necessary to define the mode of action of satraplatin. In this study, we investigate the ability of satraplatin to induce cell cycle perturbation, clonogenicity loss and apoptosis in colorectal cancer (CRC) cells.

Methods:

CRC cells were treated with satraplatin, and the effects of satraplatin on apoptosis and the cell cycle were evaluated by flow cytometry. Western blot analysis was used to investigate the effects of satraplatin on cell cycle and apoptosis-related proteins. RT-qPCR was used to evaluate p53-related mRNA modulation.

Results:

Satraplatin induced an accumulation of CRC cells predominantly in the G₂/M phase. Increased p53 protein expression was observed in the p53 wild-type HCT116 and LoVo cells together with p21^waf1/cip1 protein up-regulation. However, p21^waf1/cip1 protein accumulation was not observed in the p53 mutant HCT15, HT29, and WiDr cells, even when p53 protein expression was compromised, suggesting that the cell cycle perturbation is p53-p21^waf1/cip1 independent. Following a candidate approach, we found an elevated expression of 14-3-3σ protein levels in CRC cells, which was independent of the status of p53, further supporting the role of satraplatin in the perturbation of the G₂/M cell cycle phase. Moreover, satraplatin treatment induced apoptosis along with Bcl-2 protein down-regulation and abrogated the clonogenic formation of CRC cells in vitro.

Conclusion:

Collectively, our data suggest that satraplatin induces apoptosis in CRC cells, which is preceded by cell cycle arrest at G₂/M due to the effect of 14-3-3σ and in a p53-p21^waf1/cip1–independent manner. Taken together, these findings highlight the potential use of satraplatin for CRC treatment.     

绒毛膜促性腺激素治疗对隐睾患儿生殖细胞p53和细胞周期调节基因表达的影响

目的观察绒毛膜促性腺激素(HCG)治疗对隐睾患儿生殖细胞肿瘤抑制蛋白p53和细胞周期调节基因(p21waf/cip1)表达的影响,探讨隐睾患儿HCG治疗是否损伤睾丸生殖细胞及其机制。方法 60名隐睾患儿随机分为单纯手术组(30例)和HCG加手术组(30例),单纯手术组患儿只接受手术治疗,HCG加手术组患儿于手术前给予HCG治疗。两组患儿术后均采用RT-PCR技术检测睾丸生殖细胞p53和p21waf/cip1 mRNA表达水平。结果与单纯手术组相比,HCG加手术组p53mRNA表达增加,且两者差异有统计学意义(P<0.05);但p21waf/cip1mRNA的表达HCG加手术组较单纯手术组下降,且差异有统计学意义(P<0.05)。结论隐睾患儿给予HCG干预后,可使p53基因表达增加,p21waf/cip1基因表达下降,进而可能会导致睾丸生殖细胞增殖障碍。     

Physalis angulata induced G2/M phase arrest in human breast cancer cells.

Physalis angulata (PA) is employed in herbal medicine around the world. It is used to treat diabetes, hepatitis, asthma and malaria in Taiwan. We have evaluated PA as a cancer chemopreventive agent in vitro by studying the role of PA in regulation of proliferation, cell cycle and apoptosis in human breast cancer cell lines. PA inhibited cell proliferation and induced G2/M arrest and apoptosis in human breast cancer MAD-MB 231 and MCF-7 cell lines. In this study, under treatment with various concentrations of PA in MDA-MB 231 cell line, we checked mRNA levels for cyclin A and cyclin B1 and the protein levels of cyclin A and cyclin B1, Cdc2 (cyclin-dependent kinases), p21(waf1/cip1) and P27(Kip1) (cyclin-dependent kinase inhibitors), Cdc25C, Chk2 and Wee1 kinase (cyclin-dependent kinase relative factors) in cell cycle G2/M phase. From those results, we determined that PA arrests MDA-MB 231 cells at the G2/M phase by (i) inhibiting synthesis or stability of mRNA and their downstream protein levels of cyclin A and cyclin B1, (ii) increasing p21(waf1/cip1) and P27(kip1) levels, (iii) increasing Chk2, thus causing an increase in Cdc25C phosphorylation/inactivation and inducing a decrease in Cdc2 levels and an increase in Wee1 level. According to the results obtained, PA appears to possess anticarcinogenic properties; these results suggest that the effect of PA on the levels of phosphorylated/inactivated Cdc25C are mediated by Chk2 activation, at least in part, via p21(waf1/cip1) and P27(kip1) cyclin-dependent kinase inhibitors pathway to arrest cells at G2/M phase in breast cancer carcinoma cells.     

Genistein-induced neuronal apoptosis and G2/M cell cycle arrest is associated with MDC1 up-regulation and PLK1 down-regulation

The aim of the present study is to investigate the effect of genistein on human neuroblastoma SK-N-MC cells. MTT proliferation assay, LDH cytotoxicity assay, flow cytometric analysis, real-time quantitative RT-PCR and western blotting were used to investigate the effect of genistein on cell survival, cellular toxicity, cell cycle progression, and mRNA and protein alterations of selected DNA damage-, cell cycle- and apoptosis-related genes in SK-N-MC cells. Genistein suppressed cell proliferation, increased LDH release and modulated cell cycle distribution through accumulation of cells at G2/M- and S-phase and sub-G0 (cell death) with a concurrent decrease of cells at G0/G1 phase. Genistein increased the MDC1 (Mediator of DNA damage Checkpoint protein 1), p53, p21(waf1/cip1), Cdc2 and Bax mRNA levels in a dose-dependent manner. However, PLK1 (Polo-Like Kinase 1) and Cyclin B1 mRNAs were down-regulated after genistein treatment. Furthermore, Genistein did not alter Chk2 (Checkpoint Kinase 2), Bcl-2 and Cdc25C mRNA levels. On western blotting analyses; genistein increased the protein level of MDC1, p53, p21(waf1/cip1), and Bax in a dose-dependent manner. Genistein also increased the phosphorylation of Chk2 and Cdc25C at Thr-68 and Ser-216, respectively. In addition, consistently with PLK1 down-regulation, the phosphorylation of Cdc25C at Ser-198 was markedly decreased after genistein treatment. Additionally, Chk2, Cdc25C, Cyclin B1, p-Cyclin B1 (Ser-147), and Cdc2 as well as Bcl-2 proteins were down-regulated after genistein treatment. Altogether, these results suggest for the first time the involvement of MDC1 up-regulation after genistein treatment in DNA damage-induced Chk2 activation- and PLK1 down-regulation-mediated apoptosis and cell cycle checkpoint pathways.     

Effects of genistein on cell proliferation and cell cycle arrest in nonneoplastic human mammary epithelial cells: involvement of Cdc2, p21(waf/cip1), p27(kip1), and Cdc25C expression

Genistein, a soy isoflavone, has been reported to inhibit the multiplication of numerous neoplastic cells, including those in the breast. However, there is limited information on the effect of genistein on nonneoplastic human breast cells. In the present studies, genistein inhibited proliferation of, and DNA synthesis in, the nonneoplastic human mammary epithelial cell line MCF-10F with an IC(50) of approximately 19-22 microM, and caused a reversible G2/M block in cell cycle progression. Genistein treatment (45 microM) increased the phosphorylation of Cdc2 by 3-fold, decreased the activity of Cdc2 by 70% after 8 hr, and by 24 hr reduced the expression of Cdc2 by 70%. In addition, genistein enhanced the expression of the cell cycle inhibitor p21(waf/cip1) by 10- to 15-fold, increased p21(waf/cip1) association with Cdc2 by 2-fold, and increased the expression of the tumor suppressor p53 by 2.8-fold. Genistein did not alter the expression of p27(kip1) significantly. Furthermore, genistein inhibited the expression of the cell cycle-associated phosphatase Cdc25C by 80%. From these results, we conclude that genistein inhibits the growth of nonneoplastic MCF-10F human breast cells by preventing the G2/M phase transition, induces the expression of the cell cycle inhibitor p21(waf/cip1) as well as its interaction with Cdc2, and inhibits the activity of Cdc2 in a phosphorylation-related manner. Down-regulation of the cell cycle-associated phosphatase Cdc25C combined with up-regulation of p21(waf/cip1) expression appear to be important mechanisms by which genistein decreases Cdc2 kinase activity and causes G2 cell cycle arrest.     

Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to Adriamycin

Although the primary response to Adriamycin (doxorubicin) in p53 mutant MDA-MB231 and p53 null MCF-7/E6 breast tumor cells is apoptotic cell death, the residual surviving population appears to be in a state of senescence, based on cell morphology, beta galactosidase staining, induction of p21^waf1/cip1 and down regulation of cdc2/cdk1. Suppression of apoptosis in MDA-MB231 and MCF-7/E6 cells treated with Adriamycin using the broad spectrum caspase inhibitor, zvad-Fmk, results in substantial induction of autophagy. Overall sensitivity to Adriamycin, measured by clonogenic survival, is not altered in the cells undergoing autophagy, consistent with autophagy contributing to cell death in response to Adriamycin. The free radical scavengers, glutathione and N-acetyl cysteine attenuate the accelerated senescence response to Adriamycin in MCF-7 cells as well as in MDA-MB231 and MCF-7/E6 cells, but protect primarily the MCF-7 cells, indicating that reactive oxygen is unlikely to be directly responsible for Adriamycin toxicity in breast tumor cells. Expression of caspase 3 or induced expression of c-myc in MCF-7 cells fails to abrogate accelerated senescence induced by Adriamycin. Taken together, these studies suggest that accelerated senescence induced by Adriamycin is similar in cells with wild type p53 and in cells lacking functional p53 with regard to the upregulation of p21^waf1/cip1, down regulation of cdc2 and the involvement of reactive oxygen species. Furthermore, accelerated senescence, autophagy and apoptosis all appear to be effective in suppressing self-renewal capacity in breast tumor cells exposed to Adriamycin.     

凝血酶诱发人支气管上皮细胞恶性转化

目的探讨凝血酶对人类支气管上皮细胞的致癌性和转化涉及的相关机制。方法用75nmo·lL-1凝血酶持续刺激人类乳头状瘤-18永生化的人支气管上皮细胞(BEP2D)70d,观察不同代龄细胞的血清抗性、软琼脂克隆形成率,流式细胞术观察细胞周期改变,逆转录-聚合酶链反应检测凝血酶受体-蛋白酶活化受体(PAR1),c-myc和p21~(cip1)基因表达水平,蛋白质免疫印迹检测C-MYC和P21~(cip1)蛋白表达变化,鉴定细胞的恶性转化表型。结果经传代培养,凝血酶处理的第10代BEP2D细胞血清抗性增强,接种效率提高,第20代细胞呈非贴壁依赖性生长,软琼脂克隆形成率升高达(0.461±0.009)%。流式细胞术结果显示,第22代细胞处于S期比例为55.63%。凝血酶转化第25代、第27代细胞PAR1基因、c-myc基因及C-MYC蛋白表达水平上调。相反,p21~(cip1)基因和P21~(cip1)蛋白表达减弱。凝血酶处理的第20代细胞已具有较为稳定的转化表型和细胞生物学特性,可能是具有裸小鼠致瘤型的恶性转化细胞。每升2×104抗凝血酶单位水蛭素能够拮抗凝血酶对BEP2D细胞的恶性转化作用。结论凝血酶对人支气管上皮细胞具有恶性转化作用,c-myc基因转录和蛋白表达活化与p21~(cip1)失活在这一过程中发挥关键作用。     

Genistein arrests hepatoma cells at G2/M phase: involvement of ATM activation and upregulation of p21waf1/cip1 and Wee1

Genistein, a soy isoflavone, has a wide range of biological actions that suggest it may be of use in cancer prevention. We have recently reported that it arrests hepatoma cells at G2/M phase and inhibits Cdc2 kinase activity. In the present study, we examined the signaling pathway by which genistein modulates Cdc2 kinase activity in HepG2 cells and leads to G2/M arrest, and found that it caused an increase in both Cdc2 phosphorylation and expression of the Cdc2-active kinase, Wee1. Genistein also enhanced the expression of the cell cycle inhibitor, p21waf1/cip1, which interacts with Cdc2. Furthermore, phosphorylation/inactivation of Cdc25C phosphatase, which dephosphorylates/activates Cdc2, was increased. Genistein enhanced the activity of the checkpoint kinase, Chk2, which phosphorylates/inactivates Cdc25C, induced accumulation of p53, and activated the ataxia-telangiectasia-mutated (ATM) gene. Caffeine, an ATM kinase inhibitor, inhibited these effects of genistein on Chk2, p53, and p21waf1/cip1. These findings suggest that the effect of genistein on G2/M arrest in HepG2 cells is partly due to ATM-dependent Chk2 activation, an increase in Cdc2 phosphorylation/inactivation as a result of induction of Wee1 expression, and a decrease in Cdc2 activity as a result of induction of p21waf1/cip1 expression.     

氧化苦参碱对人食管癌细胞株Eca109增殖及凋亡的影响

目的:观察氧化苦参碱(Oxy)对食管癌细胞株Eca109的抑制增殖及诱导凋亡作用。方法:培养食管癌细胞株(Eca109),以MTT比色法、生长曲线、及流式细胞术测定Oxy对食管癌细胞株Eca109抑制增殖及诱导凋亡的作用。免疫沉淀法并ERK活性试剂盒测定ERK活性;免疫印记法测定p-ERK1/2、Cyclin D1、p21waf/cip1、Bax及Bcl-2表达。结果:MTT实验及生长曲线显示Oxy明显抑制Eca109细胞增殖,流式细胞术显示Oxy可诱导Eca109细胞凋亡;免疫沉淀法显示Oxy可抑制ERK活性,免疫印记法显示Oxy抑制p-ERK1/2、Cyclin D1及Bcl-2表达,同时上调p21waf/cip1和Bax表达,Bax/Bcl-2比值增加。结论:氧化苦参碱可以抑制食管癌细胞株Eca109增殖,机制与影响ERK及下游CyclinD1、p21waf/cip1表达有关;其诱导凋亡途径与上调Bax表达,降低Bcl-2表达有关。     

Topoisomerase I inhibitor (camptothecin)-induced apoptosis in human gastric cancer cells and the role of wild-type p53 in the enhancement of its cytotoxicity

Camptothecin (CPT), a human topoisomerase I inhibitor, blocks DNA replication in human cancer cells. It represents a promising new class of chemotherapeutic agents with broad anti-tumor activity. However, its effect on gastric cancer cells remains unknown. We examined cell growth, apoptosis and cell cycle phase distribution in gastric cancer cells by exposing these cells to CPT for up to 72 h. Cell viability was determined by the Trypan blue exclusion assay. Cell cycle phase distribution and apoptosis were measured using flow cytometry, fluorescence microscopy and DNA ladder assay. Exposure of exponentially growing gastric AGS cancer cells to CPT induced time-dependent apoptosis and growth inhibition. Serum starvation-synchronized AGS cells (about 60% cells in G0/G1 phase) showed similar cellular responses. Analysis of cell cycle phase distribution of AGS cells treated with CPT for up to 72 h showed no obvious differences compared to untreated control cells. Although the induction of apoptosis was noticed in gastric cancer cell lines both with and without p53, cells lacking p53 showed less apoptosis compared to those cell lines possessing p53. Our data show that CPT is capable of inducing gastric cancer cell growth inhibition and apoptosis. Wild-type p53 may enhance the cytotoxicity of CPT against gastric carcinoma.     

蟾蜍灵对肝癌细胞SMMC 7721的细胞毒作用及生长相关基因表达的影响

为探讨蟾蜍灵的抗癌作用机理 ,以肝癌SMMC772 1细胞为靶细胞 ,应用噻唑蓝还原法检测细胞毒作用 ,双荧光染色法和DNA电泳技术检测细胞凋亡与坏死 ;免疫组织化学方法检测细胞生长相关基因p2 1waf1/cip1和增殖细胞核抗原 (PCNA)蛋白表达 .结果表明 ,0 .0 1μmol·L- 1及以上浓度蟾蜍灵对SMMC772 1细胞具有显著细胞毒作用 ,形态学和DNA片段化检测证实蟾蜍灵诱导的细胞死亡以凋亡为主 ;p2 1waf1/cip1在蟾蜍灵诱导下表达上调 ,同时PCNA的表达下降 ,两者呈负相关 (P <0 .0 1) .提示蟾蜍灵通过上调p2 1waf1/cip1表达 ,下调PCNA表达 ,从而抑制肝癌细胞生长     

Utility of peptide–protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide*

Abstract: We used a N‐biotinylated peptide analog of the C‐terminal domain of the tumor suppressor protein, p21^cip1/waf1 to elucidate peptide/protein interacting partners. The C‐terminal domain of p21^cip1/waf1 protein spanning 141–160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell‐cycle control. This C‐terminal 20‐mer efficiently extracts PCNA in the presence of a variety of N‐ or C‐terminally attached affinity tags. Using difference silver stained 2D gels combined with in‐gel tryptic digests, we identified the difference spots using MALDI‐TOF mass spectrometry‐based peptide mass fingerprinting followed by a database search using profound against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor‐1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion‐trap LC‐MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using sequest confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90α, HSP40 and T‐complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21^cip1/waf1 protein. The use of N‐biotinylated peptide derived from the C‐terminal domain of p21^cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

Gambogic acid induces growth inhibition and differentiation via upregulation of p21waf1/cip1 expression in acute myeloid leukemia cells

Gambogic acid (GA) is the major active ingredient of gamboges, a brownish to orange resin product from Garcinia hanburyi tree in Southeast Asia. This compound exhibits anti-cancer effect on solid tumors. In this study, we investigated the effects of GA on the growth and differentiation of acute myeloid leukemia cells by growth-inhibition detection, morphological changes observation, nitroblue tetrazolium reduction, and the expression of the relative cell-surface differentiation markers. The results showed that GA could inhibit cell growth and promote differentiation in U937 and HL-60 cells. In addition, GA upregulated the expression of p21waf1/cip1 in the two cell lines. Finally, downregulating the p21waf1/cip1 expression with small interfering RNA partially blocked GA-induced cell growth inhibition and differentiation. These results of this study revealed that GA may be used as one of the investigational drugs for acute myeloid leukemia.     

Iron-induced oxidative damage and apoptosis in cerebellar granule cells: attenuation by tetramethylpyrazine and ferulic acid

Tetramethylpyrazine and ferulic acid are two active ingredients of a Chinese herbal medicine Ligusticum wallichi Franchat. In the present investigation, iron-induced oxidative neuronal damage and the protective effects of tetramethylpyrazine and ferulic acid against this induction were studied in primary cultures of rat cerebellar granule cells. When neurons were treated with 200 microM of FeSO(4) for 1 h, lipid peroxidation in neurons increased time dependently, as measured with the thiobarbituric acid assay. Thirty-six hours after iron treatment, the cell viability decreased to 43.6% and the percentage of apoptotic cells increased to 50.6%. Transmission electron microscopic examination showed a disrupted nuclear envelope and condensed chromatin in iron-treated neurons. Analysis of DNA extracted from iron-treated cells by agarose gel electrophoresis showed the typical "ladder pattern", which indicated the formation of mono- and oligonucleosomes. After iron treatment, caspase 3 activity increased significantly, as measured in a fluoregenic assay. The results above suggested that iron treatment triggered oxidative stress and apoptosis in neurons. Western blot revealed that iron treatment up-regulated the apoptosis-related gene p53 as well as its effector gene p21(waf1/cip1). Pretreatment of the cells with 100 microM of tetramethylpyrazine or ferulic acid effectively decreased the activation of caspase 3 as well as the expression of p53 and p21(waf1/cip1), and attenuated iron-induced oxidative damage and apoptosis. The results suggest that tetramethylpyrazine and ferulic acid might be used as preventive agents against neuronal diseases associated with oxidative stress.     

Influence of p53 and caspase 3 activity on cell death and senescence in response to methotrexate in the breast tumor cell

The influence of p53 function and caspase 3 activity on the capacity of the antifolate, methotrexate, to promote senescence arrest and apoptotic cell death was investigated in breast tumor cells. In p53 wild-type, but caspase 3 deficient MCF-7 breast tumor cells, death of approximately 40% of the cell population was observed immediately after acute exposure to 10 microM methotrexate (the IC80 value for a 2 h drug exposure). There was no evidence of either DNA fragmentation, a sub G0 population or morphological alterations indicative of apoptosis; however, PARP cleavage was detected. Cell death was succeeded by growth arrest for at least 72 h--where arrest was characterized by expression of the senescence marker, beta-galactosidase. The response to methotrexate in MCF-7/E6 cells with attenuated p53 function was also primarily growth arrest--but lacking characteristics of senescence. In contrast, MCF-7 cells which expressed caspase 3 demonstrated a gradual and continuous loss of cell viability and unequivocal morphological evidence of apoptosis. DNA fragmentation indicative of apoptosis was also detected after exposure to methotrexate in p53 mutant MDA-MB231 breast tumor cells which also express caspase 3. Methotrexate-induced both p53 and p21waf1/cip1 in MCF-7 cells within 6 h; however, no significant DNA strand breakage was evident before 18 h, suggesting that the induction of p53 reflects a response to cellular stress other than DNA damage, such as nucleotide depletion. Overall, these studies suggest that the nature of the cellular response to methotrexate depends, in large part, on p53 and caspase function. p53 appears to be required for methotrexate-induced senescence, but not apoptosis, caspase 3 is required for DNA fragmentation and the morphological changes associated with apoptosis, while neither p53 nor caspase 3 are required for methotrexate-induced growth arrest. Furthermore, the senescence phenotype may occur in the absence of direct DNA damage.     

Cadmium affects genes involved in growth regulation during two-stage transformation of Balb/3T3 cells

Cadmium (Cd), a carcinogenic metal in human and rodents, has been shown to transform cells in vitro. However, the carcinogenic mechanisms of Cd as a mutagen and/or promoter are not well clarified. We already reported that CdCl2 in a range of 1.5 approximately 360 ng/ml enhanced transformation of Balb/3T3 A31 cells induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 microg/ml) in a dose-dependent manner (Fang et al., Toxicol. In Vitro 15(3) (2001a) 51-7). In previous study, we observed that Cd stimulated cell proliferation on MNNG-initiated cells through inactivation of p53 and p27 and increase of proliferating cell nuclear antigen (PCNA) expression after 24 h treatment (Fang et al., Toxicology 163 (2001b) 175-84). The aim of this study is to further elucidate the long-term effect of Cd in terms of cell cycle control gene expressions during the promotion stage of in vitro two-stage transformation. For the purpose, we determined the expression levels of the genes involved in growth regulation, such as p53, p27, c-myc, mdm2, cyclins D1 and B1, CDK4, and PCNA in the cells treated with Cd for 14 days after MNNG-initiation. In MNNG+CdCl2 group, cells apparently expressed cellular tumor antigen p53 mRNA, but did not express the wild-type p53 protein; the protein and mRNA levels of p27 were reduced apparently in the cells of MNNG+CdCl2 group compared to the cells of control and MNNG group. In addition, the protein levels of cyclin D1, CDK4, PCNA, c-myc, and mdm2, and cyclin B1 mRNA level were higher in MNNG+CdCl2 group than control and MNNG group. Together with previous data (Fang et al., Toxicology 163 (2001b) 175-84), our results indicated that during the transformation process of MNNG-treated cells, Cd may activate oncogenes such as c-myc, mdm2, and cellular tumor antigen p53, inhibit the tumor suppressor genes such as wild-type p53 and p27, and consequently accelerate the proliferation of initiated cells. This work firstly demonstrates that Cd affects the genes involved in growth regulation on initiated cells during the promotion stage of in vitro cell transformation.