Hydroxyurea (HU) can increase fetal hemoglobin (HbF) in sickle cell anemia (HbSS). To identify determinants of the HbF response, we studied 150 HU-treated patients grouped by quartiles of change in HbF from baseline to 2 years. Half of the HU-assigned patients had long-term increments in HbF. In the top two quartiles, HbF increased to 18.1% and 8.8%. These patients had the highest baseline neutrophil and reticulocyte counts, and largest treatment-associated decrements in these counts. In the lower two quartiles, 2-year HbF levels (4.2% and 3.9%) and blood counts changed little from baseline. In the highest HbF response quartile, myelosuppression developed in less than 6 months, compliance was best, and final doses of HU were 15 to 22.5 mg/kg. All four quartiles had substantial increases of F cells in the first year. This was maintained for 2 years only in the top three quartiles. Leukocyte and reticulocyte counts decreased initially in all quartiles, but drifted back toward baseline levels in the lowest HbF response quartile. Initial HbF level and phenotype of the F-cell production (FCP) locus were not associated with HbF response, but absence of a Central African Republic (CAR) haplotype was. Bone marrow ability to withstand HU treatment may be important for sustained HbF increases during HU treatment of HbSS.

Proteins containing "cold shock" domains belong to the most evolutionarily conserved family of nucleic acid-binding proteins known among bacteria, plants, and animals. One of these proteins, YB-1, is widely expressed throughout development and has been implicated as a cell survival factor that regulates the transcription and/or translation of many cellular growth and death-related genes. For these reasons, YB-1 deficiency has been predicted to be incompatible with cell survival. However, the majority of YB-1 ؊/؊ embryos develop normally up to embryonic day 13.5 (E13.5). After E13.5, YB-1 ؊/؊ embryos exhibit severe growth retardation and progressive mortality, revealing a nonredundant role of YB-1 in late embryonic development. Fibroblasts derived from YB-1 ؊/؊ embryos displayed a normal rate of protein synthesis and minimal alterations in the transcriptome and proteome but demonstrated reduced abilities to respond to oxidative, genotoxic, and oncogene-induced stresses. YB-1 ؊/؊ cells under oxidative stress expressed high levels of the G 1 -specific CDK inhibitors p16Ink4a and p21Cip1 and senesced prematurely; this defect was corrected by knocking down CDK inhibitor levels with specific small interfering RNAs. These data suggest that YB-1 normally represses the transcription of CDK inhibitors, making it an important component of the cellular stress response signaling pathway.Over the past decade, an increasing number of multifunctional regulatory factors that control gene expression at both the transcriptional and posttranscriptional levels have been described. Human YB-1 and its vertebrate homologues are one example. YB-1 belongs to a large family of proteins that contain a conserved nucleic acid-binding domain termed the cold shock domain (CSD), which shares about 40% amino acid sequence identity with bacterial cold shock proteins. Among the CSD family of proteins, YB-1 and its orthologues constitute an exceptionally conserved subfamily that contains nearly identical amino acid sequences for the whole molecule (Ͼ98% identity between the human and mouse proteins). While several members were initially identified as DNA-binding proteins that interacted specifically with a conserved cis-regulatory Ybox element, YB-1 proteins have since been shown to bind avidly in vitro to a wide variety of forms of nucleic acids, including pyrimidine-rich single-stranded DNA, triplex/singlestranded H-DNA, damaged DNA, and RNA (references 18 and 36 and references therein).Based on its broad nucleic acid-binding properties, a myriad of cellular functions have been ascribed to YB-1. In mammalian cells, YB-1 has been shown to shuttle between the nuclear and cytoplasmic compartments (reference 25 and references therein). Within the nucleus, YB-1 interacts directly with other DNA-and pre-mRNA-binding proteins such as p53, AP2, CTCF, hnRNP K, and SRp30c (for a complete list, see a recent review [20]). To date, YB-1 has been implicated in nuclear activities such as chromatin remodeling, transcriptional regulation, DNA repair, and pre-mRNA...

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Although the functions of granzyme A (GzmA) and GzmB are well-defined, a number of orphan granzymes of unknown function are also expressed in cytotoxic lymphocytes. Previously, we showed that a targeted loss-of-function mutation for GzmB was associated with reduced expression of several downstream orphan granzyme genes in the lymphokine-activated killer cell compartment. To determine whether this was caused by the retained phosphoglycerate kinase I gene promoter (PGK-neo) cassette in the GzmB gene, we retargeted the GzmB gene with a LoxP-flanked PGK-neo cassette, then removed the cassette in embryonic stem cells by transiently expressing Cre recombinase. Mice homozygous for the GzmB null mutation containing the PGK-neo cassette (GzmB−/−/+PGK-neo) displayed reduced expression of the closely linked GzmC and F genes in their MLR-derived CTLs and lymphokine-activated killer cells; removal of the PGK-neo cassette (GzmB−/−/ΔPGK-neo) restored the expression of both genes. Cytotoxic lymphocytes derived from mice with the retained PGK-neo cassette (GzmB−/−/+PGK-neo) had a more severe cytotoxic defect than those deficient for GzmB only (GzmB−/−/ΔPGK-neo). Similarly, GzmB−/−/+PGK-neo mice displayed a defect in the allogeneic clearance of P815 tumor cells, whereas GzmB−/−/ΔPGK-neo mice did not. These results suggest that the retained PGK-neo cassette in the GzmB gene causes a knockdown of GzmC and F expression, and also suggest that these granzymes are relevant for the function of cytotoxic lymphocytes in vitro and in vivo.

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Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed.

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Small GTPase Ras homologue enriched in brain (RHEB) binds and activates the key metabolic regulator mTORC1, which has an important role in cancer cells, but the role of RHEB in cancer pathogenesis has not been shown. By performing a meta-analysis of published cancer cytogenetic and transcriptome databases, we defined a gain of chromosome 7q36.1-q36.3 containing the RHEB locus, an overexpression of RHEB mRNA in several different carcinoma histotypes, and an association between RHEB upregulation and poor prognosis in breast and head and neck cancers. To model gain of function in epithelial malignancy, we targeted Rheb expression to murine basal keratinocytes of transgenic mice at levels similar to those that occur in human squamous cancer cell lines. Juvenile transgenic epidermis displayed constitutive mTORC1 pathway activation, elevated cyclin D1 protein, and diffuse skin hyperplasia. Skin tumors subsequently developed with concomitant stromal angio-inflammatory foci, evidencing induction of an epidermal hypoxia-inducible factor-1 transcriptional program, and paracrine feed-forward activation of the interleukin-6-signal transducer and activator of transcription 3 pathway. Rheb-induced tumor persistence and neoplastic molecular alterations were mTORC1 dependent. Rheb markedly sensitized transgenic epidermis to squamous carcinoma induction following a single dose of Ras-activating carcinogen 7,12-dimethylbenz(a)anthracene. Our findings offer direct evidence that RHEB facilitates multistage carcinogenesis through induction of multiple oncogenic mechanisms, perhaps contributing to the poor prognosis of patients with cancers overexpressing RHEB. Cancer Res; 70(8); 3287-98. ©2010 AACR.