Human WRN and BLM genes are members of the conserved RECQ helicase family. Mutations in these genes are associated with Werner and Bloom syndromes. WRN and BLM proteins are implicated in DNA replication, recombination, repair, telomere maintenance, and transcription. Using microfluidics-assisted display of DNA for replication track analysis (ma-RTA), we show that WRN and BLM contribute additively to normal replication fork progression, and non-additively, in a RAD51-dependent pathway, to resumption of replication after arrest by hydroxyurea (HU), a replication-stalling drug. WRN but not BLM is required to support fork progression after HU. Resumption of replication by forks may be necessary but is not sufficient for timely completion of the cell cycle after HU arrest, as depletion of WRN or BLM compromises fork recovery to a similar degree, but only BLM depletion leads to extensive delay of cell division after HU, as well as more pronounced chromatin bridging. Finally, we show that recovery from HU includes apparent removal of some of the DNA that was synthesized immediately after release from HU, a novel phenomenon that we refer to as nascent strand processing, NSP.

Loss-of-function mutations in the human RecQ helicase genes WRN and BLM respectively cause the genetic instability/cancer predisposition syndromes Werner syndrome and Bloom syndrome. To identify common and unique functions of WRN and BLM, we systematically analyzed cell proliferation, cell survival, and genomic damage in isogenic cell lines depleted of WRN, BLM, or both proteins. Cell proliferation and survival were assessed before and after treatment with camptothecin, cis-diamminedichloroplatinum(II), hydroxyurea, or 5-fluorouracil. Genomic damage was assessed, before and after replication arrest, by γ-H2AX staining, which was quantified at the single-cell level by flow cytometry. Cell proliferation was affected strongly by the extent of WRN and/or BLM depletion, and more strongly by BLM than by WRN depletion (P = 0.005). The proliferation of WRN/BLM-codepleted cells, in contrast, did not differ from BLM-depleted cells (P = 0.34). BLM-depleted and WRN/BLM-codepleted cells had comparably impaired survival after DNA damage, whereas WRN-depleted cells displayed a distinct pattern of sensitivity to DNA damage. BLM-depleted and WRN/BLMcodepleted cells had similar, significantly higher γ-H2AX induction levels than did WRN-depleted cells. Our results provide new information on the role of WRN and BLM in determining cell proliferation, cell survival, and genomic damage after chemotherapeutic DNA damage or replication arrest. We also provide new information on functional redundancy between WRN and BLM. These results provide a strong rationale for further developing WRN and BLM as biomarkers of tumor chemotherapeutic responsiveness. Cancer Res; 70(16); 6548-55.

Background:Renal cell carcinoma (RCC) patients treated with tyrosine kinase inhibitors (TKI) typically respond initially, but usually develop resistance to therapy. We utilised transcriptome analysis to identify gene expression changes during development of sunitinib resistance in a RCC patient-derived xenograft (PDX) model.Methods:RCC tumours were harvested during pre-treatment, response and escape phases. Direct anti-proliferative effects of sunitinib plus MEK inhibitor were assessed. Activation status (phosphorylation) of MEK1/2 and ERK1/2 was determined, myeloid-derived suppressor cells (MDSC) sub-fractions were quantitated and G-CSF was measured by ELISA.Results:During the response phase, tumours exhibited 91% reduction in volume, characterised by decreased expression of cell survival genes. After 4-week treatment, tumours developed resistance to sunitinib, associated with increased expression of pro-angiogenic and cell survival genes. During tumour escape, cellular movement, inflammatory response and immune cell trafficking genes were induced, along with intra-tumoural accumulation of MDSC. In this PDX model, either continuous treatment with sunitinib plus MEK inhibitor PD-0325901, or switching from sunitinib to PD-0325901 was effective. The combination of PD-0325901 with TKI suppressed intra-tumoural phospho-MEK1/2, phospho-ERK1/2 and MDSC.Conclusions:Continuous treatment with sunitinib alone did not maintain anti-tumour response; addition of MEK inhibitor abrogated resistance, leading to improved anti-tumour efficacy.

The experiments described in this report are an extension of previous observations that certain adrenal steroids, when added in vitro, are able to influence the ameboid migration of leucocytes (Ketchel and Favour, 1953 a). Although other investigators have noted effects on leucocytes of adrenal steroids added in vitro (Martin et al., 1954, Leahy andMorgan, 1952), the capillary tube method of measuring leucocyte migration has proved sensitive enough to permit dose-response relationships to be determined.The capillary tube method was first used by Wright (1915), and later by Hoist (1921), Ketchel and Favour (1953), and O'Neill and Favour (1955. This consists of centrifuging small capillary tubes filled with blood so that a buffy coat of leucocytes is formed at the interphase between the packed red cells and the plasma. The distance which the leucocytes migrate into the plasma can then be measured. Ketchel and Favour (1955) showed that the migration of leucocytes in a blood sample from a healthy individual tends to remain fairly constant from day to day. However, 6-or 5-fold differences may occur in the average migration rates of normal individuals. It was also noted that acute and chronic illnesses are accompanied by wide fluctuations in leucocyte migration. The major control of leucocyte migration is mediated by factors in the plasma. Fractionation of the plasma indicates that two protein fractions oppose one another in their effect--Cohn's fraction II enhances leucocyte migration, and Cohn's fraction I I I inhibits leucocyte migration. MetkodsThe details of studying leucocyte migration by the capillary tube method have been described in a previous publication (Ketchel and Favour, 1955). In the present experiments, heparinized venous blood was separated into cells and plasma by centrifugation. The cells were washed in Hanks's solution (Hanks and Wallace, 1949), and 0.2 ml. of washed cells

Consensus guidelines recommend a number of screening examinations for survivors following allogeneic hematopoietic cell transplantation (HCT) but the frequency of detecting abnormal findings is unknown. We reviewed medical records of 118 patients who had comprehensive, standardized evaluations at one year after allogeneic HCT at Fred Hutchinson Cancer Research Center/Seattle Cancer Care Alliance. Abnormal findings were common, including moderate-severe pulmonary dysfunction (16%), fasting hyperlipidemia (56%), osteopenia (52%), osteoporosis (6%), and active chronic graft-versus-host disease (64%). Recurrent malignancy (4%) and chronic graft-versus-host disease (29%) were detected in previously unsuspected cases. Only 3% of patients had no abnormal findings. We conclude that comprehensive evaluation at one year after allogeneic HCT detects a high frequency of medical problems. Longer follow-up will be required to determine whether early detection and intervention affects late morbidity and mortality.