Alternatively spliced variants of several oncogenes and tumor suppressors have been

Alternatively spliced variants of several oncogenes and tumor suppressors have been shown to be important for their tumorigenicity. Materials and Methods Ethics Statement The study involving human subjects was conducted under a protocol approved by the RPCI Institutional Review Board (CIC0215). All tissue specimens were collected from patients who provided written informed consent. Patients and Ovarian Tumor Specimens Flash frozen tissue specimens (n?=?47) were obtained from patients 6080-33-7 IC50 undergoing debulking surgery for epithelial ovarian cancer at the Roswell Park Cancer Institute (RPCI), Buffalo, NY between 1995 and 2006. Normal ovarian samples (n?=?9) were obtained from patients undergoing hysterectomies for benign conditions such as leiomyoma. Clinicopathologic information for the entire cohort, including response to chemotherapy, is maintained in 6080-33-7 IC50 a database in the Department of Gynecologic Oncology. Cell Culture Ovarian cancer cell lines SKOV3 and OVCAR3 were obtained from the American Type Culture Collection (ATCC; Manassas, VA). A2780 and A2008 cells and their cDDP-resistant counterparts A2780/CP [25] and A2008/C13 [26] cells were obtained from Dr. Steven Howell (University of California, San Diego). These cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). A non-transformed ovarian surface epithelial cell line (IOSE-385, hereafter designated as IOSE) was immortalized with the SV40 early genes [27] and obtained from Dr. Nelly Auersperg (University of British 6080-33-7 IC50 Columbia, Canada). IOSE cells were maintained in M199/MCDB 105 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 5% FBS and gentamycin (Invitrogen, Carlsbad, CA). shRNA and rescue-SRPK1 Constructs shSRPK1-1 and shSRPK1-2, encoding shRNA targeting nucleotides 288 to 308 (and and that targeting nucleotides 288 to 308 of the SRPK1 mRNA resulted in a stronger silencing effect. Figure 3 Forced inhibition of SRPK1 expression in ovarian cancer cell lines enhances sensitivity to cisplatin. Knockdown of SRPK1 Expression Decreased Cell Proliferation and Tumorigenic Potential of SKOV3 Cells Data shown in Figure 3D also indicate that cells with reduced SRPK1 expression by shRNA grew slower than the control pSM2-EV cells. They formed fewer colonies than the non-treated control group in the absence of cDDP treatment. To further test whether reducing SRPK1 expression inhibits ovarian cancer cell proliferation, the doubling time of pSM2-EV and pshSRPK1-c5 cells was compared. Cell proliferation rate was determined in exponentially growing cells by trypsinization and trypan blue dye exclusion at 24, 48, 72, and 96 hr post plating. Figure 4A shows that the doubling time of pshSRPK1-c5 is approximately 1.4 fold of the control pSM2-EV cells (31 hr vs. 22 hr). This was confirmed by MTT assay (data not shown). We next investigated the tumorigenic potential of SRPK1 using the anchorage-independent-growth (AIG) assay. SRPK1 knockdown and control cells were seeded in the soft agar and allowed to grow for 3 weeks. Figure 4B shows that both shSRPK1 clones produced fewer and smaller colonies in soft agar, suggesting a reduction in tumorigenicity. Cell motility is one of the factors that contribute to tumor cell invasion. To test whether 6080-33-7 IC50 cell migration ability is compromised in the SRPK1 knockdown cells, an cell migration (wound healing) assay was performed. Figure 4C shows that the average migration rate for shSRPK1-c5 cells (5.80.81 unit) was approximately 60% of that for the control pSM2-EV cells (9.91.5 unit). Together, our data indicate that SRPK1 contributes to 6080-33-7 IC50 cell proliferation, cell migration and tumorigenic potential of SKOV cells. Figure 4 SRPK-knockdown ovarian cancer cells exhibit reduced cell proliferation, anchorage-independent growth and migration ability. Knockdown of SRPK1 Expression Reduced Phosphorylation of Certain SR Proteins and MAPK/AKT Proteins SR proteins are the direct targets of SRPK1 [15]. The phosphorylation pattern of these SR splicing factors is expected to be affected in SRPK1 knockdown cells. To test this prediction, Western blot analysis using a pan antibody recognizing a phospho-specific Rabbit polyclonal to ACE2 epitope common to multiple SR proteins [29] was performed. As expected, reduced SRPK1 expression resulted in decreased levels of phosphorylation of certain SR proteins in SKOV3 cells (Figure 5, middle panel). It is interesting to note that different.

Leave a Reply

Your email address will not be published.