p21 loss cooperates with INK4 inactivation facilitating immortalization and Bcl-2-mediated anchorage-independent growth of oncogene-transduced primary mouse fibroblasts

The INK4 and CIP cyclin-dependent kinase (Cdk) inhibitors (CKI) activate pocket protein function by suppressing Cdk4 and Cdk2, respectively. Although these inhibitors are lost in tumors, deletion of individual CKIs results in modest proliferation defects in murine models. We have evaluated cooperativity between loss of all INK4 family members (using cdk4r24c mutant alleles that confer resistant to INK4 inhibitors) and p21(Waf1/Cip1) in senescence and transformation of mouse embryo fibroblasts (MEF). We show that mutant cdk4r24c and p21 loss cooperate in pRb inactivation and MEF immortalization. Our studies suggest that cdk4r24c mediates resistance to p15(INK4B)/p16(INK4A) that accumulates over passage, whereas loss of p21 suppresses hyperoxia-induced Cdk2 inhibition and pRb dephosphorylation on MEF explantation in culture. Although cdk4r24c and p21 loss cooperate in H-ras(V12)/c-myc-induced foci formation, they are insufficient for oncogene-induced anchorage-independent growth. Interestingly, p21(-/-); cdk4r24c MEFs expressing H-ras(V12) and c-myc display detachment-induced apoptosis and are transformed by c-myc, H-ras(V12), and Bcl-2. We conclude that the INK4 family and p21 loss cooperate in promoting pRb inactivation, cell immortalization, and H-ras(V12)/c-myc-induced loss of contact inhibition. In addition, absence of pRb function renders H-ras(V12) + c-myc-transduced fibroblasts prone to apoptosis when deprived of the extracellular matrix, and oncogene-induced anchorage-independent growth of pocket protein-deficient cells requires apoptotic suppression.     

Mysterious liaisons: the relationship between c-Myc and the cell cycle.

A large body of physiological evidence shows that either upregulation or downregulation of intracellular c-Myc activity has profound consequences on cell cycle progression. Recent work suggests that c-Myc may stimulate the activity of cyclin E/cyclin-dependent kinase 2 (Cdk2) complexes and antagonize the action of the Cdk inhibitor p27KIP1. Cyclin D/Cdk4/6 complexes have also been implicated as targets of c-Myc activity. However, in spite of considerable effort, the mechanisms by which c-Myc interacts with the intrinsic cyclin/Cdk cell cycle machinery remain undefined.     

Receptor-selective retinoids inhibit the growth of normal and malignant breast cells by inducing G1 cell cycle blockade

Summary Despite advances in treatment, breast cancer continues to be the second leading cause of cancer mortality in women. Statistics suggest that while focus on treatment should continue, chemopreventive approaches should also be pursued. Previous studies have demonstrated that naturally occurring retinoids such as 9-cis retinoic acid (9cRA) can prevent breast cancer in animal models. However, these studies have also shown that these compounds are too toxic for general use. Work from our laboratory showed that an RXR-selective retinoid LGD1069 prevented tumor development in animal models of cancer with reduced toxicity as compared to an RAR-selective retinoid TTNPB. In the present study, we investigated the mechanisms by which receptor-selective retinoids inhibit the growth of normal and malignant breast cells. Our results demonstrate that the synthetic retinoids tested are as effective as 9cRA in suppressing the growth of normal human mammary epithelial cells (HMECs) and estrogen receptor-positive (ER-positive) breast cancer cells. Although the receptor-selective retinoids induce minimal amounts of apoptosis in T47D breast cancer cells, the predominant factor that leads to growth arrest is G1 cell cycle blockade. Our data indicate that this blockade results from the downregulation of Cyclin D1 and Cyclin D3, which in turn causes Rb hypophosphorylation. Non-toxic retinoids that are potent inducers of cell cycle arrest may be particularly useful for the prevention of breast cancer.     

The CDK4/6 inhibitor PD0332991 reverses epithelial dysplasia associated with abnormal activation of the cyclin-CDK-Rb pathway

Loss of normal growth control is a hallmark of cancer progression. Therefore, understanding the early mechanisms of normal growth regulation and the changes that occur during preneoplasia may provide insights of both diagnostic and therapeutic importance. Models of dysplasia that help elucidate the mechanisms responsible for disease progression are useful in highlighting potential targets for prevention. An important strategy in cancer prevention treatment programs is to reduce hyperplasia and dysplasia. This study identified abnormal upregulation of cell cycle-related proteins cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, and phosphorylated retinoblastoma protein (pRb) as mechanisms responsible for maintenance of hyperplasia and dysplasia following downregulation of the initiating viral oncoprotein Simian virus 40 (SV40) T antigen. Significantly, p53 was not required for successful reversal of hyperplasia and dysplasia. Ligand-induced activation of retinoid X receptor and PPARγ agonists attenuated cyclin D1 and CDK6 but not CDK4 or phosphorylated pRb upregulation with limited reversal of hyperplasia and dysplasia. PD0332991, an orally available CDK4/6 inhibitor, was able to prevent upregulation of cyclin D1 and CDK6 as well as CDK4 and phosphorylated pRb and this correlated with a more profound reversal of hyperplasia and dysplasia. In summary, the study distinguished CDK4 and phosphorylated pRb as targets for chemoprevention regimens targeting reversal of hyperplasia and dysplasia.     

Expression of G1 phase-related cell cycle molecules in naturally developing hepatocellular carcinoma of Long-Evans Cinnamon rats

It has been shown that a variety of cell cycle-related proteins play important roles in the process of carcinogenesis including hepatocarcinogenesis. In the present study, we evaluated mRNA and protein expression of G1 phase-related cell cycle molecules in the process of hepatocarcinogenesis, using Long-Evans Cinnamon (LEC) rats, an animal model of hepatocellular carcinoma (HCC). The expression of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6 was measured quantitatively by real-time polymerase chain reaction. Cyclin D1 mRNA expression was increased significantly in chronic hepatitis liver compared with normal liver, and then decreased in HCC and the surrounding precancerous liver of LEC rats. Levels of Cdk4 mRNA were increased significantly in HCC compared to precancerous and chronic hepatitis livers. In contrast, mRNA levels of Cdk6 did not change significantly during hepatocarcinogenesis. We also evaluated the protein levels of these G1 phase-related cell cycle molecules by Western blot analyses and confirmed similar results. Total amounts of retinoblastoma protein (pRb) in the liver did not change significantly in the process of hepatocarcinogenesis in LEC rats. However, levels of phosphorylated pRb were increased markedly in the process of hepatocarcinogenesis, and the highest in HCC compared to precancerous, chronic hepatitis and normal livers. These results indicate that cyclin D1 may be involved in the regeneration of hepatocytes rather than hepatocarcinogenesis, while Cdk4 but not Cdk6 may play an important role in the development of HCC.     

N-(4-hydroxyphenyl)retinamide induces growth arrest and apoptosis in HTLV-I-transformed cells.

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid that inhibits growth and induces apoptosis in many human cell lines. We explored the effects of HPR on human T-cell lymphotropic virus type I (HTLV-I)-positive and HTLV-I-negative malignant T-cell lines, most of which are resistant to all-trans retinoic acid. Clinically achievable concentrations of HPR caused a dramatic inhibition of cell proliferation, G(0)/G(1) arrest, and massive apoptosis in all tested malignant T cells, while no effect was observed on resting or activated normal lymphocytes. Interestingly, HTLV-I-negative cell lines were significantly more sensitive to HPR compared to HTLV-I-positive and Tax-transfected cells. In HTLV-I-negative cells only, HPR-induced apoptosis was associated with ceramide accumulation, sharp decrease in mitochondrial membrane potential, and activation of caspases 8, 9 and 3, and could be partially reverted by the caspase inhibitor z-VAD suggesting that Tax protects infected cells from ceramide accumulation and caspase-mediated apoptosis. In HTLV-I-positive cells, HPR treatment rapidly induced proteasomal-mediated degradation of p21, downregulated cyclin D(1), and upregulated bax protein levels. These findings support a potential therapeutic role for HPR in both HTLV-I-associated adult T-cell leukemia/lymphoma (ATL) and HTLV-I-negative peripheral T-cell lymphomas.     

Phosphorylation of ErbB4 on tyrosine 1056 is critical for ErbB4 coupling to inhibition of colony formation by human mammary cell lines

In many studies, ErbB4 expression in breast tumor samples correlates with a favorable patient prognosis. Similarly, ErbB4 signaling is coupled to cellular differentiation and growth arrest in a variety of model systems. However, in some studies, ErbB4 expression in breast tumor samples correlates with poor outcome. Likewise, studies using some human mammary tumor cell lines suggest that ErbB4 is coupled to malignant phenotypes. Thus, the roles that ErbB4 plays in human breast cancer are still poorly defined. Here we demonstrate that a constitutively active ErbB4 mutant (ErbB4-Q646C) inhibits colony formation on plastic by two human mammary tumor cell lines (SKBR3 and MCF7) and by the MCF10A immortalized human mammary cell line, but does not inhibit colony formation by the MDA-MB-453 and T47D human mammary tumor cell lines. ErbB4 kinase activity is necessary for ErbB4 function and phosphorylation of ErbB4 Tyr1056 is necessary and appears to be sufficient for ErbB4 function. The inhibition of colony formation by MCF10A cells is accompanied by growth arrest but not cell death. These data suggest that ErbB4 behaves as a mammary tumor suppressor and that loss of ErbB4 coupling to growth arrest may be an important event in mammary tumorigenesis.     

Altered G1 phase regulation in osteosarcoma

Alterations in the normal cell cycle lead to abnormal cell proliferation and to tumor development. To explore the role of the cyclin D/Cdk4 complex and the retinoblastoma protein (pRb) in the growth and spread of osteoblastic osteosarcoma (OS), 40 tumor samples were selected. In 17 of these cases, lung metastases occurred during follow-up. Expression of pRb, cyclin D1 and its catalytic subunit, Cdk4, was studied by immunohistochemistry and immunoblotting. As controls, non-neoplastic tissues surrounding the tumor were used. The expression level and pattern were compared to clinical outcome. Cdk4 was over-expressed in 80% of OS, independently of clinical outcome, and showed an intense and uniform distribution in tumor cells compared to normal cells. However, co-immunoprecipitation of Cdk4 with cyclin D1 revealed low levels of cyclin D/Cdk4 complex; 20 of 40 OS examined had a negative or minimal immunostaining for active pRb. The probability of relapse was significantly higher in pRb-negative than in the -positive patients (p < 0.05). The ratio of unphosphorylated/hyperphosphorylated pRb was lower in relapsed patients than in patients with no evident disease, though the difference was not statistically significant. High levels of pRb/cyclin D1 were found in all samples exhibiting functional pRb expression. Our results show that G₁ phase deregulation is involved in formation and development of OS. The expression levels of both pRb and cyclin D1 had a clear correlation with clinical outcome, suggesting that these parameters could be used as prognostic markers. Int. J. Cancer 74:518–522, 1997. © 1997 Wiley-Liss, Inc.     

Temporally distinct roles for tumor suppressor pathways in cell cycle arrest and cellular senescence in Cyclin D1-driven tumor

ABSTRACT: BACKGROUND: Cellular senescence represents a tumor suppressive response to a variety of aberrant and oncogenic insults. We have previously described a transgenic mouse model of Cyclin D1-driven senescence in pineal cells that opposes tumor progression. We now attempted to define the molecular mechanisms leading to p53 activation in this model, and to identify effectors of Cyclin D1-induced senescence. Results: Senescence evolved over a period of weeks, with initial hyperproliferation followed by cell cycle arrest due to ROS production leading to activation of a DNA damage response and the p53 pathway. Interestingly, cell cycle exit was associated with repression of the Cyclin-dependent kinase Cdk2. This was followed days later by formation of heterochromatin foci correlating with RB protein hypophosphorylation at Cdk4-dependent sites. In the absence of the Cdk4-inhibitor p18Ink4c, cell cycle exit was delayed but most cells eventually showed a senescent phenotype. However, tumors later arose from this premalignant, largely senescent lesion. We found that the p53 pathway was intact in tumors arising in a p18Ink4c-/- background, indicating that the two genes represent distinct tumor suppressor pathways. Upon tumor progression, both p18Ink4c-/- and p53-/- tumors showed increased Cdk2 expression. Inhibition of Cdk2 in cultured pre-tumorigenic and tumor cells of both backgrounds resulted in decreased proliferation and evidence of senescence. Conclusion: Our findings indicate that the p53 and the RB pathways play temporally distinct roles in senescence induction in Cyclin D1-expressing cells, and that Cdk2 inhibition plays a role in tumor suppression, and may be a useful therapeutic target.     

Removal of Cdk inhibitors through both sequestration and downregulation in zearalenone-treated MCF-7 breast cancer cells

Treatment of MCF 7 cells with the fungal estrogen zearalenone induced cyclin E-associated kinase activity transiently within 9-12 h; total cyclin-dependent kinase (Cdk) 2 activity was elevated for 24 h and beyond. This increased cyclin E/Cdk2 activity was associated with sequestration of the Cdk inhibitor p27 Cdk inhibitor 1B (p27(KIP1)) by newly formed cyclin D1/Cdk4 complexes and with downregulation of p27(KIP1) expression. The activation of cyclin A/Cdk2 activity corresponded with virtual elimination of p27(KIP1). The activity of cyclin E/Cdk2 complexes from zearalenone-treated lysates was inhibited in vitro by recombinant p27(KIP1), and this inhibition was relieved by the addition of recombinant cyclin D1/Cdk4 complexes. Thus, sequestration of p27(KIP1) by cyclin D1/Cdk4 resulted in activation of Cdk2 in vitro. Cdk inhibitory activity in lysates of zearalenone-treated cells was depleted by anti-p27(KIP1) and anti-Cdc2 interacting protein (p21(CIP1)) antibodies. Overexpression of the Cdk4/6-specific Cdk inhibitor of Cdk4 p16(INK4A) was associated with increased association of p27(KIP1) with Cdk2, concomitant with disruption of D cyclin/Cdk4 complexes. The proteasome inhibitor 2-leu-leu-leu-H aldehyde (MG-132) was relatively ineffective in inhibiting the initial, sequestration-dependent activation of cyclin E/Cdk2 yet was as effective as p16(INK4A) in inhibiting activation of cyclin A/Cdk2 later in G(1). Downregulation of p27(KIP1) proceeded in p16(INK4A)-expressing cells after zearalenone treatment, and G(1) arrest afforded by p16(INK4A) expression was reversible upon prolonged treatment with zearalenone. Zearalenone treatment of MCF-7 cells elicited expression of F-box protein S phase kinase-associated protein 2 (p45(SKP2)), a substrate-specific component of the ubiquitin-ligase complex that targets p27(KIP1) for degradation in the proteasome. These studies suggest that both sequestration of Cdk inhibitors by cyclin D1/Cdk4 complexes and downregulation of p27(KIP1) play major roles in the induction of Cdk2 activity and S phase entry elicited by estrogens in MCF-7 cells.     

细胞周期调控相关蛋白Cyclin D1、CDK 4和pRb在新疆维吾尔族妇女宫颈癌中的表达及意义

 目的 探讨细胞周期调控相关蛋白Cyclin D1、CDK 4和pRb磷酸化状态在新疆维吾尔族妇女宫颈癌（简称维族）中表达及其意义。方法 应用免疫组织化学方法检测64例维族宫颈癌及43例维族正常宫颈组织中Cyclin D1、CDK 4表达和pRb磷酸化状态。结果 Cyclin D1和CDK4在宫颈癌组中阳性率分别为70%、87%,与正常宫颈组相比,差异有统计学意义（P<0.05）,pRb磷酸化在两组中差异有统计学意义（P<0.01）。结论 Cyclin D1 与CDK4 蛋白的高表达及pRb高磷酸化可能与维族宫颈癌发生发展有关。     

The prolyl oligopeptidase inhibitor SUAM-14746 attenuates the proliferation of human breast cancer cell lines in vitro

Background

Prolyl oligopeptidase (POP, EC 3.4.1.26) is a serine peptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We previously reported that POP inhibition suppressed the growth of NB-1 human neuroblastomas cells and KATO III human gastric cancer cells. POP activity is commonly elevated in many cancers, which includes breast cancer. However, the effect of POP inhibition as a candidate breast cancer therapy is unknown.

Methods

The effects of POP inhibition and knockdown on the proliferation of cultured human estrogen receptor-positive (ER+) MCF7 and T47D, and ER-negative (ER?) MDA-MB-231 breast cancer cell lines and the MCF12A non-tumorigenic epithelial cell line were tested by analyzing their influence on cell proliferation (WST-1 assay), cell viability (trypan blue exclusion assay), and cell cycle arrest (cell cycle analysis, cell cycle regulator proteins expression).

Results

POP-specific inhibitors 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thiopropyl-thioprolinal and RNAi-mediated POP knockdown inhibited the proliferation of MCF7 cells without inducing cell death. SUAM-14746-induced growth inhibition was also observed in T47D and MDA-MB-231 cells, but not in MCF12A cells. This growth inhibition was associated with G₁ phase arrest; reduced cyclin D1 and D3, cyclin-dependent kinase 4 (CDK4), E2F1, and retinoblastoma protein (pRb) expression; and increased cyclin-dependent kinase inhibitor 1B (p27^kip1) expression. Moreover, the SUAM-14746-mediated cell cycle arrest of MCF7 cells was associated with increased pRb2/p130 protein expression and an increase in the number of cells in the quiescent G₀ state, as defined by low RNA levels.

Conclusions

SUAM-14746 inhibited breast cancer cell growth in a cytostatic manner without inducing lethality, and POP-specific inhibitors may be an effective treatment against ER+ and ER? breast cancer.

     

Reactive oxygen species mediate N-(4-hydroxyphenyl)retinamide-induced cell death in malignant T cells and are inhibited by the HTLV-I oncoprotein Tax.

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid that inhibits growth of many human tumor cells, including those resistant to natural retinoids. HPR is an effective chemopreventive agent for prostate, cervix, breast, bladder, skin and lung cancers, and has shown promise for the treatment of neuroblastomas. We have previously shown that HPR inhibits proliferation and induces apoptosis of human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia (ATL) and HTLV-I-negative malignant T cells, whereas no effect is observed on normal lymphocytes. In this report, we identified HPR-induced reactive oxygen species (ROS) generation as the key mediator of cell cycle arrest and apoptosis of malignant T cells. HPR treatment of HTLV-I-negative malignant T cells was associated with a rapid and progressive ROS accumulation. Pre-treatment with the antioxidants vitamin C and dithiothreitol inhibited ROS generation, prevented HPR-induced ceramide accumulation, cell cycle arrest, cytochrome c release, caspase-activation and apoptosis. Therefore, anti-oxidants protected malignant T cells from HPR-induced growth inhibition. The expression of the HTLV-I oncoprotein Tax abrogated HPR-induced ROS accumulation in HTLV-I-infected cells, which explains their lower sensitivity to HPR. Defining the mechanism of free radical induction by HPR may support a potential therapeutic role for this synthetic retinoid in ATL and HTLV-I-negative T-cell lymphomas.     

Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells

T47D cells represent an estrogen-responsive human ductal carcinoma cell line which expresses detectable levels of estrogen receptor (ER). We have previously shown that estradiol (E(2)) treatment of T47D cells causes an increase in the level of p53 and a concomitant phosphorylation of retinoblastoma protein (pRb). In the present study, we have analysed the expression of p53 and phosphorylation state of pRb and compared the effects of E(2) and triiodothyronine (T(3)) on these phenomena. Cells were grown in a medium containing charcoal-treated serum to deplete the levels of endogenous steroids. Upon confluency, the cells were treated with T(3) (10(-12) to 10(-7) M) for 24 h and the presence of p53 and pRb was detected by Western analysis. E(2) treatment of cells caused a 2-3-fold increase in the level of p53. Presence of T(3) in the medium caused a gradual increase in the level of p53 in a concentration-dependent manner. Under the above conditions, pRb was phosphorylated (detected as an upshift during SDS-PAGE) in the presence of E(2) and T(3). Supplementation of growth medium with T(3) (1 microM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this effect was maintained for up to 12 h. When ICI 164 384 (ICI) (1 microM), an ER antagonist, was combined with E(2) (1 nM) or T(3) (1 microM), effects of hormones on cell proliferation and hyperphosphorylation of pRb were blocked. Western analysis of p53 was supplemented with its cytolocalization by immuno-labeling using laser scanning confocal fluorescence microscopy, which revealed an ICI-sensitive increase in the abundance of p53 in hormone-treated cells. Steroid binding analysis revealed lack of competition by T(3) for the [(3)H]E(2) binding. These results indicate that T(3) regulates T47D cell cycle progression and proliferation raising the p53 level and causing hyperphosphorylation of pRb by a common mechanism involving ER and T(3) receptor (T(3)R)-mediated pathways.     

Selective in vivo and in vitro effects of a small molecule inhibitor of cyclin-dependent kinase 4

BACKGROUND: Cyclin-dependent kinase 4 (Cdk4) represents a prime target for the treatment of cancer because most human cancers are characterized by overexpression of its activating partner cyclin D1, loss of the natural Cdk4-specific inhibitor p16, or mutation(s) in Cdk4's catalytic subunit. All of these can cause deregulated cell growth, resulting in tumor formation. We sought to identify a small molecule that could inhibit the kinase activity of Cdk4 in vitro and to then ascertain the effects of that inhibitor on cell growth and tumor volume in vivo. Methods: A triaminopyrimidine derivative, CINK4 (a chemical inhibitor of Cdk4), was identified by screening for compounds that could inhibit Cdk4 enzyme activity in vitro. Kinase assays were performed on diverse human Cdks and on other kinases that were expressed in and purified from insect cells to determine the specificity of CINK4. Cell cycle effects of CINK4 on tumor and normal cells were studied by flow cytometry, and changes in phosphorylation of the retinoblastoma protein (pRb), a substrate of Cdk4, were determined by western blotting. The effect of the inhibitor on tumor growth in vivo was studied by use of tumors established through xenografts of HCT116 colon carcinoma cells in mice. Statistical tests were two-sided. RESULTS: CINK4 specifically inhibited Cdk4/cyclin D1 in vitro. It caused growth arrest in tumor cells and in normal cells and prevented pRb phosphorylation. CINK4 treatment resulted in statistically significantly (P: =.031) smaller mean tumor volumes in a mouse xenograft model. CONCLUSIONS: Like p16, the natural inhibitor of Cdk4, CINK4 inhibits Cdk4 activity in vitro and slows tumor growth in vivo. The specificity of CINK4 for Cdk4 raises the possibility that this small molecule or one with a similar structure could have therapeutic value.