p53Val135 temperature sensitive mutant suppresses growth of human breast cancer cells

Summary One common step in the malignant progression of a wide variety of human cancers seems to be inactivation of the p53 gene, via point mutation or deletion or both; or overexpression of mutated protein with dominant transforming activity. This study shows a suppressive effect of wild type p53 on the growth of human breast cancer cells. Introduction of wild type p53 versus mutant into five human breast cancer cell lines containing mutant p53 resulted in a marked reduction in colony formation. Two of these were transfected with human wt p53 expression vectors and the other three were infected with retroviruses packaging human wt p53, both showing similar reduction in the number of surviving colonies, suggesting a role for wt p53 in suppression of breast cancer cell growth. Direct evidence for growth suppression was obtained by introduction of the temperature sensitive p53Val135 into Hs578T human breast cancer cells containing a mutant p53. This murine mutant allele p53Val135 functions as an oncogene at 37° C and as a tumor suppressor at 32° C. The cell line generated was strongly growth inhibited at the restrictive temperature (31.5° C), at which temperature the suppressor form is expressed. This inhibition of proliferation was reversible upon a temperature upshift. Analysis of the cell cycle distribution shows these growth suppressed cells to be inhibited in the G1 phase of the cell cycle. Thus wt p53 may have an important role in breast cancer tumorigenesis.     

ErbB-2 signaling is involved in regulating PSA secretion in androgen-independent human prostate cancer LNCaP C-81 cells

The expression and secretion of prostate-specific antigen (PSA) are regulated by androgens in normal prostate secretory epithelial cells. In prostate cancer patients, the serum PSA level is usually elevated and cancer cells are initially responsive to androgens. However, those cancer cells become androgen-independent after androgen ablation therapy. In hormone-refractory cancer patients, even in an androgen-deprived environment, the circulation level of PSA rebounds and is constitutively elevated through a yet unknown mechanism. Tyrosine phosphorylation of ErbB-2 is involved in regulating the androgen-responsive phenotype of prostate cancer cells, and it is at least partly regulated by the cellular form of prostatic acid phosphatase (PAcP), a prostate-unique protein tyrosine phosphatase. We investigated the ErbB-2 signal pathway in androgen-independent PSA secretion. LNCaP C-81 cells, which are androgen-independent LNCaP cells lacking endogenous PAcP expression with a hypertyrosine phosphorylated ErbB-2, secreted a higher level of PSA in conditioned media than did androgen-sensitive LNCaP C-33 parental cells. A restored expression of cellular PAcP in C-81 cells was concurrent with a decrease in tyrophosphorylation of ErbB-2 and reduction of PSA secretion. Moreover, transient transfection of C-33 cells with the wild-type ErbB-2 or a constitutively active mutant of MEK1 cDNA resulted in an increased level of secreted PSA. The elevation of secreted PSA level by the forced expression of ErbB-2 was inhibited by an MEK inhibitor, PD98059. In C-81 cells, the expression of a dominant negative mutant of ErbB-2 reduced the secreted level of PSA. The inhibition of ErbB-2 or mitogen-activated protein (MAP) kinases by specific inhibitors AG879, AG825, or PD98059 led to a decrease in PSA secretion. Taken together, our data clearly indicate that the ErbB-2 signal pathway via MAP kinases (ERK1/2) is involved in regulating the secretion of PSA by androgen-independent human prostate cancer LNCaP C-81 cells in an androgen-depleted environment.     

Elevated levels of prostate-specific antigen (PSA) in prostate cancer cells expressing mutant p53 is associated with tumor metastasis

The underlying basis for rising levels of prostate-specific antigen (PSA) in prostate cancer is not fully understood, but attention has turned to the possibility that loss of normal p53 function might be directly involved. We have investigated the relationship between p53 function and PSA expression using in vitro and in vivo approaches. Three prostate cancer-derived p53 mutants (F134L, M237L, R273H) were introduced into LNCaP prostate cancer cells and stable transfectants established. Expression of mutant p53 was demonstrated by Western blot analysis, inactivation of wtp53 function, and a loss of p53-dependent responses to DNA damage induced by UV-irradiation and cisplatin. Levels of PSA mRNA and secreted protein were determined by RT-PCR and Western blotting, respectively. Serine protease activity was assessed using an esterase assay. In vivo effects of mutant p53 expression were examined after orthotopic implantation into prostates of nude mice. Expression of all p53 mutants was associated with elevated PSA mRNA and secreted PSA protein. In a representative line, mutant p53 was also associated with increased PSA protease-like activity compared with a control line expressing wildtype p53. Overall PSA levels, and PSA levels in serum from mice bearing tumors derived from cells expressing mutant p53, were increased compared with levels in mice bearing tumors derived from control cells. In addition, the tumors derived from cells with mutant p53 had increased vascularization and induced lymph node metastases. These data provide in vitro and in vivo support for the notion that p53 mutations directly contribute to increased levels of serum PSA, and are associated with more aggressive tumors.     

Down-Regulation of prostate-specific antigen expression by ligands for peroxisome proliferator-activated receptor gamma in human prostate cancer

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Recent studies found that ligand-activated PPARgamma regulated differentiation and clonal growth of several types of cancer cells, including prostate cancer, suggesting that PPARgamma could be a tumor suppressor. Troglitazone was a widely used antidiabetic drug that activates PPARgamma. Recently, we reported that this agent had antiprostate cancer effects in vitro and in vivo. In this study, we administered troglitazone for over 1.5 years to an individual with occult recurrent prostate cancer. Using the prostate-specific antigen (PSA) levels as a surrogate marker of the disease, the oral administration of troglitazone (600-800 mg/day) reduced the increase velocity of PSA levels, suggesting clinical efficacy of troglitazone in prostate cancer. PSA promoter/ enhancer reporter assays showed that the PPARgamma ligands troglitazone (10(-5) M), pioglitazone (10(-5) M), or 15-deoxy-delta12,14-prostaglandin J2 (10(-5) M) down-regulated androgen-stimulated reporter gene activity in LNCaP cells, a prostate cancer cell line. The PSA promoter contains androgen receptor response elements (AREs). Reporter gene studies showed that troglitazone inhibited androgen activation of the AREs in the PSA regulatory region. Consistent with inhibition of gene expression, 2 days of incubation of LNCaP with troglitazone dramatically suppressed PSA protein expression without suppressing AR expression, suggesting that troglitazone inhibited ARE activation by a mechanism other than down-regulation of expression of the AR. Taken together, ligands of PPARgamma may be a useful therapeutic approach for the treatment of prostate cancer and may be acting, in part, by inhibiting transactivation of androgen-responsive genes.     

Interferon regulatory factor 4 binding protein is a novel p53 target gene and suppresses cisplatin-induced apoptosis of breast cancer cells

ABSTRACT: BACKGROUND: Our previous work demonstrated that ectopic expression of interferon regulatory factor 4 binding protein (IBP) was correlated with the malignant behaviour of human breast cancer cells. The mechanisms controlling differential expression of IBP in breast cancer still remain unknown. RESULTS: To investigate the mechanism of IBP dysregulation in breast cancer, we identified IBP was a novel p53 target gene. IBP expression was negatively regulated by wild-type p53 and was p53 dependently suppressed by DNA damage agent cisplatin. Furthermore, high levels of IBP were found to decrease cisplatin-induced growth suppression and apoptotic cell death, which was associated with decreased p53 activity and imbalanced Bcl-2 family member expression. CONCLUSIONS: IBP is a novel p53 target gene which suppresses cisplatin-mediated apoptosis of breast cancer cells via negative feedback regulation of the p53 signalling pathway, suggesting IBP may serve as a target for pharmacologic intervention of breast cancer resistant to cisplatin therapy.     

Tissue prostate-specific antigen facilitates refractory prostate tumor progression via enhancing ARA70-regulated androgen receptor transactivation

Despite being well recognized as the best biomarker for prostate cancer, pathophysiologic roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histologic analyses show that the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumor specimens. By stably transfecting PSA cDNA into various prostate cancer cell lines, we found that PSA could promote the growth of androgen receptor (AR)-positive CWR22rv1 and high-passage LNCaP (hormone-refractory prostate cancer cells) but not that of AR-negative PC-3 and DU145 cells. Surprisingly, the protease activity of PSA is not crucial for PSA to stimulate growth and promote AR transactivation. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating the p53 pathway that results in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G(1) phase. In vitro colony formation assay and in vivo xenografted tumor results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that, in addition to being a biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA small interfering RNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer.     

Differential effects of naturally occurring and synthetic organoselenium compounds on biomarkers in androgen responsive and androgen independent human prostate carcinoma cells

Epidemiological studies and clinical trials show that selenium supplementation results in reduction of prostate cancer incidence; however, the form of selenium and mechanisms underlying protection remain largely unknown. Toward this end, we compared the effects of naturally occurring selenomethionine (SM) and Se-methylselenocysteine (MSC) and synthetic 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and p-xylylbis(methylselenide) p-XMS) organoselenium compounds in androgen responsive (AR) LNCaP and its androgen independent clone (AI) LNCaP C4-2 human prostate carcinoma cells on cell growth, secretion of prostate specific antigen (PSA), intracellular redox status and genomic profiles with emphasis on identifying redox sensitive genes. Both p-XSC and p-XMS reduced cell number and total protein concentration compared to control-treated AR and AI cells, while SM and MSC exhibited no effect on growth of AR and AI cells. SM, p-XSC and p-XMS but not MSC inhibited levels of secreted PSA in AR cells. SM, MSC and p-XMS increased glutathione (GSH) levels in AI LNCaP cells. By contrast, in both cell types, only p-XSC significantly decreased GSH concentrations to <50% of control suggesting either an increase in intracellular oxidative stress or a change in GSH/GSSG ratio. On the basis of RT-PCR analysis, SM and p-XSC increased p53 gene expression by 2-fold in AR cells but not in AI cells and only SM enhanced epidermal growth factor receptor in AR cells. Depending on the structure, organoselenium compounds exhibit differential effects on growth, PSA secretion, oxidative stress and selective gene responses in human prostate cancer cells and suggest the potential of developing novel organoselenium compounds as chemopreventive agents in models of human prostate cancer.     

p300 regulates androgen receptor-independent expression of prostate-specific antigen in prostate cancer cells treated chronically with interleukin-6

Prostate cancer is the most frequent non-skin cancer in men. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor has been shown to play an important role. Androgen receptor is expressed in both early and late-stage prostate cancer. Also, androgen-regulated pathways are thought to be active as evidenced by elevated levels of prostate-specific antigen (PSA). In addition, several androgen receptor coactivators and cytokines are involved in prostate cancer progression. In this regard, we have shown previously that the coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), a cytokine involved in late-stage prostate cancer. In this study, we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronically with IL-6. We found that p300 but not CREB-binding protein induced activation of PSA in these cells and that the histone acetyltransferase activity of p300 was critical. This effect was independent of the presence of androgens or antiandrogens. Moreover, we found markedly reduced levels of androgen receptor in these cells and p300 transfection did not affect those levels, suggesting that the p300 effect on PSA could be bypassing the androgen receptor. Transfection with exogenous androgen receptor showed minimal response of PSA to androgens but higher response to p300. We found similar effects of p300 on the androgen response element III, which mediates the androgen receptor-dependent activation of PSA. Finally, we showed that p300 alone regulates expression of the endogenous PSA gene in the IL-6-treated cells. These findings reveal a new insight in the progression of prostate cancer, suggesting that coactivators, such as p300, play more important roles in late-stage prostate cancer, and could regulate androgen-dependent genes in the absence or with very low levels of androgen receptor.     

PrLZ expression is associated with the progression of prostate cancer LNCaP cells

PrLZ is a novel recent isolated gene and specific expression in prostate tissues. PrLZ expression was specifically elevated in prostate embryonic tissues and androgen independent prostate cancer cells, suggesting it might be association with the embryonic development and malignancy progression. However, the function and mechanism of PrLZ during the progression of prostate cancer remain blurred. Our present studies showed PrLZ expression might enhance the proliferation and invasion capability in vitro and also increase the tumorigenicity in situ prostate cancer animal model, which is indicated PrLZ expression contributed to the malignancy progression of prostate cancer. In addition, PrLZ also might up regulate androgen receptor (AR) expression and increase the PSA expression, a putative downstream target gene of AR, which indicated PrLZ mediated the malignancy progression of prostate cancer was associated with androgen signals.     

野生型p14ARF基因转染对人肺癌细胞生长抑制的这实验研究

目的 探讨外源性p14ARF基因能否对体内外生长的肺癌细胞起抑制增殖的作用。方法 选择4株具有不同基因背景的肺癌细胞系,转染人的野生型p14ARF基因。通过RT-PCR、免疫组化及Westernblot杂交检测,筛选出同时表达p14ARF mRNA及蛋白的阳性克隆。以母细胞及转染空载体细胞为对照,比较它们的增殖、细胞周期分布、裸鼠体内成瘤性以及形态学上的差异。结果 三株具有野生型p53基因的肺癌细胞在转染p14ARF基因后,细胞周期被显著阻滞于G1或G1/G2期,增殖受到抑制。结论 外源性野生型p14ARF基因转染能抑制人肺癌细胞的增殖,p14ARF基因是人体肺癌基因治疗的理想侯选基因。     

Co-transduction of Apaf-1 and caspase-9 highly enhances p53-mediated apoptosis in gliomas

Mutation of the p53 gene plays a critical role in the development of cancer and response to cancer therapy. To analyze the mechanism of cancer development and to improve cancer therapy, it is important to assess which genes are downstream components of p53 in cancers, and whether the expression levels of these genes affect p53-mediated apoptosis. In this study, we transduced the wild type p53 gene along with the Apaf-1 and caspase-9 genes via adenovirus vectors into U251 and U-373MG glioma cells harbouring a mutated p53, and evaluated the degree of apoptosis. Co-induction of Apaf-1 and caspase-9 genes highly enhanced p53-mediated apoptosis in glioma cells. Induction of wild type p53 enhanced the expression levels of Bax, p21/WAF1, and Fas protein. To determine which gene is activated by wild type p53 induction and, in turn, activates Apaf-1 and caspase-9, we transduced the Bax, p21/WAF1 or Fas gene via adenovirus vector to U251 cells to achieve a similar expression level as that induced by the Adv for p53 in U251 cells. U251 cells transduced with Fas concomitant with the Apaf-1 and caspase-9 genes underwent drastic apoptosis. This suggests that induction of wild type p53 upregulates Fas, which in turn may play a role in the activation of Apaf-1 and caspase-9. These results are important for analyzing the mechanism of tumour development and for predicting the therapeutic effect of p53 replacement gene therapy in a particular patient.     

The serine/threonine protein kinase, p90 ribosomal S6 kinase, is an important regulator of prostate cancer cell proliferation

An increase in the activity of mitogen-activated protein kinase (MAPK) has been correlated with the progression of prostate cancer to advanced disease in humans. The serine/threonine protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of MAPK but its role in prostate cancer has not previously been examined. Increasing RSK isoform 2 (RSK2) levels in the human prostate cancer line, LNCaP, enhanced prostate-specific antigen (PSA) expression, an important diagnostic marker for prostate cancer, whereas inhibiting RSK activity using a RSK-specific inhibitor, 3Ac-SL0101, decreased PSA expression. The RSK2 regulation of PSA expression occurred via a mechanism involving both RSK2 kinase activity and its ability to associate with the coactivator, p300. RNA interference of the androgen receptor (AR) showed that the AR was important in the RSK2-mediated increase in PSA expression. RSK levels are higher in approximately 50% of human prostate cancers compared with normal prostate tissue, which suggests that increased RSK levels may participate in the rise in PSA expression that occurs in prostate cancer. Furthermore, 3Ac-SL0101 inhibited proliferation of the LNCaP line and the androgen-independent human prostate cancer line, PC-3. These results suggest that proliferation of some prostate cancer cells is dependent on RSK activity and support the hypothesis that RSK may be an important chemotherapeutic target for prostate cancer.     

Function and therapeutic implication of C-CAM cell-adhesion molecule in prostate cancer

Human neoplasms are often caused by cumulative alterations in oncogenes and tumor-suppressor genes. By identifying the early genetic changes involved in tumorigenesis, one can develop strategies to prevent and detect cancers at early stages, when treatment is most effective. C-CAM1, a cell-adhesion molecule (CAM) isoform (I), was recently shown to play a critical role in prostate cancer initiation and progression. Loss of C-CAM1 expression occurs early in the development of prostate cancer, suggesting that C-CAM1 may help maintain the differentiated state of the prostate epithelium. Reintroduction of C-CAM1 into cancer cells can reverse their cancerous growth. Thus, the C-CAM1 molecule itself or drugs that increase C-CAM1 expression are promising agents for prostate cancer treatment. The mechanisms by which C-CAM1 suppresses tumorigenesis are different from those of p53 and Rb. Therefore, C-CAM1 therapy is a new form of prostate cancer treatment. To exploit C-CAM1's therapeutic potential, a human C-CAM1 adenovirus expression vector (Ad-hu-C-CAM1) has been used to treat prostate tumor xenografts in nude mice. The preliminary results have shown great promise. In addition, while C-CAM gene therapy may have immediate application in prostate cancer treatment, the knowledge to be learned from mechanistic studies of C-CAM1-mediated tumor suppression may also help us design better strategies for prevention and treatment for prostate cancer.     

Expression of Androgen Receptor Is Negatively Regulated By p53

Increased expression of androgen receptor (AR) in prostate cancer (PC) is associated with transition to androgen independence. Because the progression of PC to advanced stages is often associated with the loss of p53 function, we tested whether the p53 could regulate the expression of AR gene. Here we report that p53 negatively regulates the expression of AR in prostate epithelial cells (PrECs). We found that in LNCaP human prostate cancer cells that express the wild-type p53 and AR and in human normal PrECs, the activation of p53 by genotoxic stress or by inhibition of p53 nuclear export downregulated the expression of AR. Furthermore, forced expression of p53 in LNCaP cells decreased the expression of AR. Conversely, knockdown of p53 expression in LNCaP cells increased the AR expression. Consistent with the negative regulation of AR expression by p53, the p53-null HCT116 cells expressed higher levels of AR compared with the isogenic HCT116 cells that express the wildtype p53. Moreover, we noted that in etoposide treated LNCaP cells p53 bound to the promoter region of the AR gene, which contains a potential p53 DNA-binding consensus sequence, in chromatin immunoprecipitation assays. Together, our observations provide support for the idea that the loss of p53 function in prostate cancer cells contributes to increased expression of AR.