According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.
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-2(EK/+)) and in human metastatic renal cell carcinoma tissue. Signaling associated with cytoplasmic mislocalization of p27 in renal cancer is relatively unknown. Renal tumors derived from 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-treated Tsc-2(EK/+) rats, and null for tuberin, display elevated nuclear and cytosolic p27, with parallel increases in cytosolic cyclin D1 levels. Similar changes are observed in TGHQ-transformed renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), which, in addition to the cytoplasmic mislocalization of p27 and cyclin D1, exhibit high ERK, B-Raf, and Raf-1 kinase activity. Renal tumor xenografts, derived from subcutaneous injection of QTRRE cells into nude mice, also display increases in cytosolic mislocalization of p27 and cyclin D1. Dibutyryl cAMP and/or phosphodiesterase inhibitors (PIs; pentoxifylline or theophylline) increase Rap1B activation, B-Raf kinase activity, and cytosolic p27/cyclin D1 protein levels in QTRRE cells. Inhibition of Raf kinases with either sorafenib or B-Raf small interfering RNA (siRNA) caused a mitogen-activated protein kinase-mediated downregulation of p27. Moreover, decreases in cyclin D1 were also associated with p27 siRNA knockdown in QTRRE cells. Finally, theophylline-mediated increases in p27 and cyclin D1 were attenuated by sorafenib, which modulated Raf/MEK/ERK signaling. Collectively, these data suggest that the cAMP/Rap1B/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 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)
Cyclin D1 is a key regulator of cell cycle progression and a therapeutic endpoint for cancer treatment. The human prostate cancer cell lines, LNCaP (PTEN‐) and DU145 (PTEN+), were used to examine the differential regulation of cyclin D1 as a determinant of drug sensitivity to amuvatinib, a receptor tyrosine kinase inhibitor, and erlotinib, an epidermal growth factor inhibitor. 1D and 2D Western blot analysis revealed that combination drug treatment of LNCaP cells caused a decrease in 4EBP1 pSer65, pThr70, and pThr37/46, with concomitant decreases in cyclin D1 levels. Such combination treatment, however, did not modulate pERK status. Moreover, single drug treatment with amuvatinib, but not erlotinib, decreased p‐4EBP1 and cyclin D1 levels. In DU‐145 cells, combination drug treatment had no effect on p‐4EBP1 status, but decreased p‐ERK and cyclin D1 levels. Additionally, single drug treatment with erlotinib, but not amuvatinib, caused a loss of p‐ERK and cyclin D1. The data reveal the differential modulation of cyclin D1, and its upstream regulators, in prostate cancer cell lines by amuvatinib and erlotinib. Such differential responses likely contribute to specific anti‐tumor agent efficacy. An appreciation of factors that determine the cell‐dependent modulation of cyclin D1 will assist in the selection of appropriate biomarkers for directing patient‐targeted therapy. Grant Funding Source: Supported by Ventana/Roche, P30 ES006694
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