Cyclin D1 is required for progression from G1 into S phase of the cell cycle. Over‐expression of cyclin D1 causes an increase in cell cycle progression and proliferation. Canonical cyclin D1 degradation is initiated by phosphorylation of Thr286 by GSK‐3β. QTRRE (rodent) and ACHN (human) renal cell carcinoma (RCC) cell lines exhibit elevated levels of cyclin D1. Pentoxifylline (PTX) is used as an adjunct in chemotherapy to treat cachexia. We report that PTX causes a time‐ and dose‐dependent decrease in cyclin D1 levels and G1 cell cycle arrest in QTRRE and ACHN cells. RT‐PCR analysis showed no significant changes in cyclin D1 mRNA. However, PTX’s ability to decrease cyclin D1 was prevented by the proteasome inhibitor, MG‐132. Inhibition of GSK‐3β with LiCl in the presence of PTX failed to rescue cyclin D1 levels. PTX increased pSer9 on GSK‐3β, indicative of an inhibition of GSK‐3β activity. Following siRNA knockdown of GSK‐3β, PTX retained the ability to decrease cyclin D1 levels. Moreover, PTX treatment in the presence of MG‐132 revealed no increase in pThr286 compared to control. Our data indicate that PTX initiates GSK‐3β‐independent proteasomal degradation of cyclin D1 and arrests the RCC cells in the G1 phase. Because our findings demonstrate a novel anti‐cancer property of PTX, its use as an adjuvant therapy in RCC treatment should be further explored. Grant Funding Source: Supported by ES006694, AstraZeneca Studentship, T32ES016652
Cyclin D1 is required for cells to progress from the G1 phase into the S phase of the cell cycle. Several tumors display elevations in cyclin D1, concomitant with increased cell cycle progression and proliferation. QTRRE (rodent) and ACHN (human) cell models of renal cell carcinoma display elevated cyclin D1 protein levels. Pentoxifylline (PTX), a competitive non‐specific phosphodiesterase inhibitor, has found recent use as an adjunct in chemotherapy for patients to help treat cachexia and capillary leak syndrome. We report that PTX causes a time‐ (1–24 hr) and dose‐dependent (35 μM −3.5 mM) decrease in cyclin D1 protein levels in both cell lines. PTX's ability to decrease cyclin D1 in QTRRE and ACHN was abolished in the presence of a proteasome inhibitor (MG‐132, 10μM) as well as a GSK‐3β inhibitor (VI, 1μM). Consistent with decreases in cyclin D1, flow cytometric analysis revealed that QTRRE and ACHN cells treated for 24hr with PTX undergo enhanced cell cycle arrest in the G1 phase (170 & 140%, respectively). Collectively the data suggest that PTX decreases cyclin D1 protein levels by stimulating GSK‐3β‐induced proteasomal degradation, which promotes G1 phase cell cycle arrest. Moreover, because our findings reveal a novel anti‐cancer chemotherapeutic property of PTX, the utility of PTX as an adjuvant therapy in the treatment of cancer should be further explored. (AstraZeneca Studentship, T32ES016652, P30ES006694)
The loss of tuberin, the tuberous sclerosis‐2 (Tsc‐2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc‐2EK/+), and has been recently observed in human renal cell carcinoma tissue. Signaling associated with the expression and cytoplasmic mislocalization of p27 is relatively unknown in renal tumor development. TGHQ transformed renal epithelial cells derived from Tsc‐2EK/+ rats (QTRRE cells), that form renal tumors in nude mice, display cytoplasmic mislocalization of p27 and cyclin D1, and also exhibit high ERK, B‐Raf and Raf‐1 kinase activity. Dibutyryl cAMP or phosphodiesterase inhibitors ([PIs]; pentoxifylline or theophylline) cause an increase in cytosolic p27/cyclin D1 protein levels in QTRRE cells. Additionally, treatment of QTRRE cells with both PIs revealed concomitant decreases in p‐p27 T187 and in its E3 ubiquitin ligase skp1; proteosome inhibition resulted in increased p27/cyclin D1 protein levels. Inhibition of Raf kinases with either sorafenib or B‐Raf siRNA resulted in MAPK down‐regulation of p27. Moreover, an associated decrease in cyclin D1 was observed following p27 siRNA knockdown in QTRRE cells. Collectively these data suggest that the cAMP/B‐Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27‐cyclin D1 in tuberous sclerosis‐gene regulated‐renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27‐cyclin D1 cytosolic stabilization during renal tumor formation.
Cyclin D1 is required for cells to progress from G1 to S phase of the cell cycle. Mutation or over‐expression of cyclin D1 leads to uncontrolled proliferation. QTRRE (rat) and ACHN (human) renal cancer cells possess elevated levels of cyclin D1. Pentoxifylline (PTX), a non‐specific phosphodiesterase inhibitor, induces a time‐(0–24 hr) and dose‐(35 μM‐3.5 mM) dependent decrease in cyclin D1 levels in both cell models. QTRRE and ACHN cells treated with PTX (24 hr) undergo cell cycle arrest in the G1 phase (170 & 140%, respectively). While RT‐PCR showed no change in cyclin D1 transcription after PTX treatment (0–24hr), a decrease of cyclin D1 t1/2 was observed, suggesting an increase in protein degradation. Furthermore, PTX‐induced decrease of cyclin D1 was abolished in the presence of a proteasome inhibitor (MG‐132, 10μM) as well as GSK‐3β inhibitors (LiCl, 20mM and VI, 10uM). MS proteomics analysis following cyclin D1 antibody pull‐down revealed that cyclin D1 associates with proteasomal subunit α type 5 and polyubiquitins B & C. The data suggest that PTX decreases cyclin D1 levels by stimulating GSK‐3β‐induced proteasomal degradation, which promotes G1 phase cell cycle arrest. Moreover, because our findings reveal a novel anti‐cancer chemotherapeutic property of PTX, the utility of PTX as an adjuvant therapy in the treatment of cancer should be further explored. (AstraZeneca Studentship, T32ES016652, P30ES006694)
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