VMY-1-103 is a novel CDK inhibitor that disrupts chromosome organization and delays metaphase progression in medulloblastoma cells

Abstract

Medulloblastoma is the most prevalent of childhood brain malignancies, constituting 25% of childhood brain tumors. Craniospinal radiotherapy is a standard of care, followed by a 12mo regimen of multi-agent chemotherapy. For children less than 3 y of age, irradiation is avoided due to its destructive effects on the developing nervous system. Long-term prognosis is worst for these youngest children and more effective treatment strategies with a better therapeutic index are needed. VMY-1-103, a novel dansylated analog of purvalanol B, was previously shown to inhibit cell cycle progression and proliferation in prostate and breast cancer cells more effectively than purvalanol B. In the current study, we have identified new mechanisms of action by which VMY-1-103 affected cellular proliferation in medulloblastoma cells. VMY-1-103, but not purvalanol B, significantly decreased the proportion of cells in S phase and increased the proportion of cells in G(2)/M. VMY-1-103 increased the sub G(1) fraction of apoptotic cells, induced PARP and caspase-3 cleavage and increased the levels of the Death Receptors DR4 and DR5, Bax and Bad while decreasing the number of viable cells, all supporting apoptosis as a mechanism of cell death. p21(CIP1/WAF1) levels were greatly suppressed. Importantly, we found that while both VMY and flavopiridol inhibited intracellular CDK1 catalytic activity, VMY-1-103 was unique in its ability to severely disrupt the mitotic spindle apparatus significantly delaying metaphase and disrupting mitosis. Our data suggest that VMY-1-103 possesses unique antiproliferative capabilities and that this compound may form the basis of a new candidate drug to treat medulloblastoma.

Figure 1

Effects of VMY on cell cycle progression in human medulloblastoma cells. The human medulloblastoma cell lines (A), DAOY or (B) D556 were treated for 18 h with either LY294002 (LY), purvalanol B (PVB) or VMY at the concentrations shown. Cells were harvested and both DNA fragmentation (subG₁) and the cell cycle profile were measured by flow cytometry. Data are average ± standard deviation of n ≥ three separate experiments vs. DMSO.

Figure 2

Effects of VMY on DAOY cell proliferation. (A) DAOY cells were treated for 18 h with either DMSO, the dansyl group alone or VMY-1-103 at the concentrations shown. Cells were harvested and cell viability assessed by trypan blue dye exclusion on >300 cells. Data are average ± standard deviation of n ≥ three separate experiments. (B) apoptosis proteome arrays performed on extracts from DAOY cells were treated for 18 h with either DMSO or VMY at 30 uM. Fold change in protein abundance vs. DMSO are shown as Ave ± deviation of duplicate samples from n = two separate experiments. A representative proteomic array is shown at top. (C) Representative protein gel blot (n ≥ 3) performed on DAOY cells treated for 18 h with either PVB or VMY at the concentrations shown. (D) In vitro CDK1-kinase assays performed on DAOY cell extracts treated as marked. Data are percent inhibition in substrate phosphorylation vs. vehicle control for n = two experiments. Flavo, Flavopiridol; Cl- capase-3, cleaved caspase-3; PARP_FL, full length PARP; PARP_L, 89 kD fragment of cleaved PARP; Noc, nocodazole; Veh, vehicle.

Figure 3

VMY-induced disruption of mitosis in DAOY cells. Live cell imaging was performed using stably transfected DAOY/GFP-H2B cells. (A) The percent of cells that successfully completed mitosis within 6 h following release from nocodazole block. (B) Metaphase transit time. (C) Examples of transit times of cells that successfully progressed through metaphase. Data are average percent change (+ SD, n > 250 cells, two separate experiments). Arrows, significant changes in mitotic progression in VMY vs. Flavo (flavopiridol) treated cells.

Figure 4

VMY delays progression through mitosis. DAOY/GFP-H2B cells were released from nocodazole block and followed by live-cell video microscopy. Time of progression from (A) prophase to metaphase and (B) metaphase to anaphase. *p ≤ 0.05, **p ≤ 0.01.

Figure 5

Effects of VMY and flavopiridol on Mitosis in D556 cells. Stable GFP-H2B/D556 cells were treated with nocodazole for 18 h. The nocodazole was removed and the cells were treated with the compounds as shown and followed by live cell imaging. (A) average, n = four experiments. (B) Representative live cell images. **p < 0.01. arrows, significant changes in mitotic progression in VMY vs. Flavo (flavopiridol) treated cells.

Figure 6

Merged fluorescent imaging of DAOY cells. Cells were treated DMSO or VMY (30 um) for 1 h. (A and B) Cells were stained with phospho-histone H3 (yellow) and Aurora Kinase A antibodies (red) as well as with DAPI (blue) and phalloidin for F-actin (green). (C and D) Cells were stained with phospho-Wee1 (yellow) and Aurora Kinase A antibodies (red) as well as with DAPI (blue) and phalloidin for F-actin (green). The Wee1/Aurora A merge is in orange. PM, prometaphase; M, metaphase; A, anaphase; T, telophase. Arrows, trailing or misaligned chromosomes, *, mislocalized Wee1, Aurora Kinase A or phospho-histone H3.