Er during the transformation process and play a key role in
Er during the transformation process and play a key role in tumorigenesis. Previously, we have cloned a mutant variant of the RPS2 ribosomal protein, termed PCADM-1, and shown that RPS2 was over expressed in malignant prostate cell lines and in human prostate cancer (PCa) [1]. PCADM-1 was over-expressed in human PCa and not found in benign (BPH), high grade prostatic intraepithelial neoplasia (HGPIN), or seminal vesicle (SV) tissue. Likewise, the normal RPS2 gene was found to be over-expressed by malignant prostate lines (i.e. PC-3 ML and LNCaP cells), and by early stage prostate cancer cell lines (HGPIN, CPTX-1532). The data suggest that PCADM-1 and/or RPS2 might be novel bio-markers and excellent prognostic indicators for human prostate cancer. More importantly, PCADM-1 or RPS2 might be novel therapeutic targets for treating the disease. In this paper, we have examined the importance of the RPS2 gene for proliferation and survival of malignant and normal prostate cell lines in vitro and in vivo. We have developed a ‘ribozyme-like’ oligonucleotide, DNAZYM1P, which specifically targets RPS2 and found that DNAZYM-1P treatment of PC-3ML, LNCaP, and CPTX1532 cells induced a significant increase in cellular apoptosis and death (i.e. > 95 after 48 hr). Mouse tumor modeling studies further revealed that DNAZYM-1P delivered locally or systemically, eradicated primary and metastatic tumors of PC-3ML cells in SCID mice. Moreimportantly, treatment dramatically increased mice disease free survival rates by 100 . For the first time, we have convincingly demonstrated that tumors which over express the RPS2 protein can be eradicated with a DNAZYM-1P targeting this gene.MethodsCell cultures LNCaP, DU145, CRW22R1 and mouse 3T3 fibroblasts were obtained from ATCC (Bethesda, MD) and grown according to their instructions. PC-3 ML cells were maintained in DMEM plus 10 fetal bovine serum according to published methods [5]. ��-Amanitin biological activity CPTX-1532 and NPTX-1532 cells were derived from malignant and normal tissue of the same human prostate tissue, respectively [6]. BPH-1 cells [7] were a gift from Donna Peehl (Stanford Univ.). CPTX-1532, NPTX-1532, and BPH-1 cells were each immortalized with human papillomavirus serotype 16 [8]. IBC-10a [9] cells were primary ‘intermediate basal cell’ cultures derived from a Gleason score 6 prostate cancers by our lab. IBC-10a cells were subsequently immortalized with hTERT (courtesy of Johng Rhim, Bethesda, MD). The IBC-10a cells were also transfected with a pBABE-c-myc puromycin vector (courtesy of Dr. Sell, Drexel Univ., Philadelphia, PA)(the pBABE vector was purchased from Clonetics Inc., Boston, MA)) and stable clones selected for 2 weeks with 2 ug/ml puromycin. The CPTX-1532 and NPTX-1532, BPH-1, and IBC-10a were maintained at low passage (< 10) in Keratinocyte serum free media (SFM) (Life Technologies, Inc., Grand Island, NY) containing 5 ng/mL epidermal growth factor, 50 g/ mL bovine pituitary extract, plus 100 units/mL penicillin G sodium and 100 g/mL streptomycin sulfate. Cells were cultured at 37 in a humidified atmosphere of 95 air and 5 CO2. In experimental studies, the cells were harvested using a 1:10 dilution of 10X Trypsin-EDTA (Invitrogen, Grand Island, N.Y.) in PBS, pH 7.4 and washed three times with fresh keratinocyte PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27864321 SFM, counted with a hemacytometer, and plated in keratinocyte SFM o.n. prior to starting experimental treatments. Cell growth assays were carried out using MTS reagents according to methods of the man.