and has received compensation from these companies in the form of stock; A.R.K. is a research collaborator of Ionis Pharmaceuticals and has received royalty income from Ionis through his employer, Cold Spring Harbor Laboratory. O.A.-W. has served as a consultant for H3 Biomedicine, Foundation Medicine Inc., Merck, and Janssen; O.A.-W. has received personal speaking fees from Daiichi Sankyo. O.A.-W. has received prior research funding from H3 Biomedicine unrelated to the current manuscript. D.I., R.K.B. and O.A.-W. are inventors on a provisional patent application (patent number FHCC.P0044US.P) applied for by Fred Hutchinson Cancer Research Center on the role of reactivating BRD9 expression in cancer by modulating aberrant BRD9 splicing in SF3B1 mutant cells.

The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.⌬ and tip1.⌬ disrupted linear growth, corrupting peg1 + did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 + manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170.[Keywords: CLASP; dynein; S. pombe; fission yeast; CLIP170; EB1] Supplemental material is available at http://www.genesdev.org.

Deregulation of D-type cyclin-dependent kinases (CDK4 and 6) is widely observed in various human cancers, illustrating their importance in cell cycle control. Like other cyclin-dependent kinases (CDKs), assembly with cyclins is the most critical step for activation of CDK4/ 6. As previously reported elsewhere, we observed that the level of cyclinD1-CDK4 complex and its associated kinase activity were significantly low in asynchronously proliferating mouse embryo fibroblasts lacking both p21 Cip1 and p27 Kip1 (p21/p27-null MEFs). These evidences imply that p21 Cip1 and p27 Kip1 CDK inhibitors are 'essential activators' of cyclin D-kinases. We, however, discovered here that both the assembly and activation of cyclin D1-CDK4 complex occur when quiescent p21/p27-null MEFs were stimulated to re-enter the cell cycle. This mitogen-induced cyclin D1-kinase activity was blocked by overexpression of p16 INK4a and resulted in the inhibition of S phase entry in p21/p27-null MEFs. Furthermore, ectopic expression of p34 SEI-1 , a mitogen-induced CDK4 binding protein, increased the levels of active cyclinD1-CDK4 complex in asynchronously proliferating p21/p27-null MEFs. Together, our results suggest that there are several independent ways to stimulate the assembly of cyclin D1-CDK4 kinases. Although p21 Cip1 and p27 Kip1 play a role in this process, our results demonstrate that additional mechanisms must occur in G0 to S phase transition.

Summary A study was made of the relatonship between the intrinsic radiosensifivity of human cervical tumours and the expression of the DNA repair enzyme human apurnic/apyrmidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both measurements were obtained on pretreatment biopsy material. All 25 tumours examined showed positive staining for HAP1, but there was heterogeneity in the level of expression both within and between tumours. The average coefficients of variation for intra-and intertumour heterogeneity were 620o and 82% respectively. There was a moderate but significant positive correlation between the levels of HAP1 expression and SF2 (r= 0.60, P = 0.002). Hence, this study shows that there is some relationship between intrinsic radiosensitivity and expression of a DNA repair enzyme in cervical carcinomas. The resutts suggest that this type of approach may be useful in the development of rapid predictive tests of tumour radiosensitivity.