ABSTRACT
PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.
Subject(s)
Phosphoric Diester Hydrolases/metabolism , Prostatic Neoplasms/metabolism , Pyrophosphatases/metabolism , Androgen Antagonists/pharmacology , Anilides/pharmacology , Animals , Cell Line, Tumor , DNA, Antisense/genetics , Disease Progression , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , NIH 3T3 Cells , Neoplasms, Hormone-Dependent/genetics , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Nitriles/pharmacology , Oncogene Protein v-akt/metabolism , Phosphoric Diester Hydrolases/biosynthesis , Phosphoric Diester Hydrolases/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Pyrophosphatases/biosynthesis , Pyrophosphatases/genetics , Signal Transduction , Tosyl Compounds/pharmacology , TransfectionABSTRACT
Human prostate and colon gene-1 (PC-1, also known as PrLZ) is an androgen-regulated, prostate tissue and prostate cancer cells specifically expressed novel gene. The increased expression of PC-1 gene appears to promote prostate cancer cells androgen-dependent (AD) and androgen-independent (AI) growth. To clone and investigate the expression and regulation elements of PC-1 gene may provide insight into the function of PC-1 and develop a new promoter that targets therapeutic genes to the AD and AI prostate cancer cells. The goal of the present study is cloning and characterization of the PC-1 promoter. A series of luciferase constructs that contain various fragments of the PC-1 5'-genomic region were transfected into human prostate cancer cells for promoter transactivation analysis. 5' deletion analysis identified the -1579 bp promoter region was required for the maximal proximal promoter activity; two transcriptional suppression and a positive regulatory region were identified; -4939 bp promoter fragment of the PC-1 gene retained the characteristic of prostate cancer-specific expression and exhibited higher transcription activity than PSA-6 kb promoter in the medium supplemented with steroid-depleted FBS. An androgen response element (ARE) was located in between -345 and -359 bp of the PC-1 5'-untranslated region relative to the translation initiation site. Thus, our studies not only provide molecular basis of PC-1 transcription regulation, but also define a new regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer in the prostate cancer gene therapy.
Subject(s)
Phosphoric Diester Hydrolases/genetics , Promoter Regions, Genetic/genetics , Prostatic Neoplasms/genetics , Pyrophosphatases/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcriptional Activation/genetics , Base Sequence , Cell Line, Tumor , Humans , Male , Molecular Sequence DataABSTRACT
OBJECTIVE: To study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2. METHODS: Recombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells. RESULTS: PC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells. CONCLUSION: RNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.
Subject(s)
Phosphoric Diester Hydrolases/biosynthesis , Prostatic Neoplasms/genetics , Pyrophosphatases/biosynthesis , RNA Interference , Cell Line, Tumor , Down-Regulation , Gene Expression , Humans , Male , Phosphoric Diester Hydrolases/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Pyrophosphatases/genetics , RNA, Messenger/genetics , TransfectionABSTRACT
OBJECTIVE: To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene. METHODS: Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression. RESULTS: NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice. CONCLUSION: Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.
Subject(s)
Cell Transformation, Neoplastic , Genes, Neoplasm/physiology , Neoplasm Proteins/biosynthesis , Animals , Cell Line, Transformed , Gene Expression , Mice , Mice, Inbred BALB C , Mice, Nude , NIH 3T3 Cells , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Random Allocation , TransfectionABSTRACT
Phosphorus deficiency results in cluster root formation and increased organic acid exudation. The regulatory mechanisms for these processes, however, are not yet clear. In the present study, influences of 6-BA (6-benzyl aminopurine) on cluster root formation, exudation of citrate and malate and their concentrations in the root clusters of P-deficient white lupin plants were studied by using non-destructive localized collection method and high-performance liquid chromatography (HPLC) technique. The results showed that application of exogenous 6-BA to P-deficient plants did not influence plant growth and P distribution within the plant (Tables 2,3), whereas the cluster root formation (Fig. 1, Table 1) and organic acid exudation (Table 4) were inhibited. The inhibitory effects could be reversed by omitting 6-BA from the growth medium, and even some stimulatory effects was observed, when lower concentration of 6-BA (10(-8) mol/L) was applied (Fig. 1, Tables 1 and 4). The inhibitory effects of higher concentration of 6-BA (10(-7) mol/L) were not reversible. (Table 4). Treatment with 6-BA also had some influence on organic acid concentration in the tissue of cluster roots (Table 5). The possible reasons for the effects on cluster root formation and organic acid exudation by 6-BA are discussed.
