Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4

Lee, Jennifer; Shieh, Jae‐Hung; Zhang, Jianxuan; Liu, Liren; Zhang, Yue; Eom, Jae Yong; Morrone, Giovanni; Moore, Malcolm A.S.; Zhou, Pengbo

doi:10.1182/blood-2012-09-455204

Cited by 37 publications

(36 citation statements)

References 31 publications

(45 reference statements)

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“…The cell lines were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 units/mL streptomycin. The human primary CD34+ AML cells were purified using MACS CD34 MicroBeads (Miltenyl Biotec), and co-cultured with mitomycin-treated HS-5 human stromal cells (17). The peripheral blood mononuclear cells were isolated using ficoll density centrifugation (GE lifetechology).…”

Section: Methodsmentioning

confidence: 99%

“…The cells were washed in ice-cold PBS and fixed in 70% ethanol. BrdU uptake was measured by flow cytometry as described (17,18), using a FITC-anti-BrdUrd (Roche Diagnostics, Pleasanton, CA) monoclonal antibody. Subsequently, cells were stained with 0.05 mg/mL propidium iodide (Sigma-Aldrich) and 0.1% RNase (Invitrogen) for 30 minutes at room temperature, and subjected to analysis by flow cytometry.…”

Section: Methodsmentioning

confidence: 99%

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CDK4/6 Inhibitor PD 0332991 Sensitizes Acute Myeloid Leukemia to Cytarabine-Mediated Cytotoxicity

et al. 2015

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Cyclin-dependent kinase (CDK)4 and CDK6 are frequently overexpressed or hyperactivated in human cancers. Targeting CDK4/CDK6 in combination with cytotoxic killing therefore represents a rational approach to cancer therapy. By selective inhibition of CDK4/CDK6 with PD 0332991, which leads to early G1 arrest and synchronous S phase entry upon release of the G1 block, we have developed a novel strategy to prime acute myeloid leukemia (AML) cells for cytotoxic killing by cytarabine (Ara-C). This sensitization is achieved in part through enrichment of S-phase cells, which maximizes the AML populations for Ara-C incorporation into replicating DNA to elicit DNA damage. Moreover, PD 0332991 trigged apoptosis of AML cells through inhibition of the homeobox (HOX)A9 oncogene expression, reducing the transcription of its target PIM1. Reduced PIM1 synthesis attenuates PIM1-mediated phosphorylation of the pro-apoptotic BAD and activates BAD-dependent apoptosis. In vivo, timely inhibition of CDK4/CDK6 by PD 0332991 and release profoundly suppresses tumor growth in response to reduced doses of Ara-C in a xenograft AML model. Collectively, these data suggest selective and reversible inhibition of CDK4/CDK6 as an effective means to enhance Ara-C killing of AML cells at reduced doses, which has implications for the treatment of elderly AML patients who are unable to tolerate high dose Ara-C therapy.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

CDK4/6 Inhibitor PD 0332991 Sensitizes Acute Myeloid Leukemia to Cytarabine-Mediated Cytotoxicity

et al. 2015

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“…For the His-tagged ubiquitin-expressing plasmid ubiquitination assay, 80% confluent HEK293 cells were transiently transfected with 2 g of His 6 -tagged ubiquitin-expressing plasmid (11,12), HA-CUL4B (generated by Dr. Zhou's lab), and/or MYC-INSL6 (purchased pENTR223-INSL6 plasmid from Harvard PlasmID Database, then subcloned INSL6 ORF into pCS2-Myc vector in Dr. Zhou's lab) as specified, and treated with 20 M MG132 2 h prior to harvesting. Trypsinized cells were pelleted and washed in PBS, and 1/20 of the cell aliquots were boiled in 2ϫ SDS-PAGE buffer for Western blot analysis.…”

Section: Cul4bmentioning

confidence: 99%

Cell Autonomous and Nonautonomous Function of CUL4B in Mouse Spermatogenesis

Yin¹,

Liu

Yang

et al. 2016

Journal of Biological Chemistry

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CUL4B ubiquitin ligase belongs to the cullin-RING ubiquitin ligase family. Although sharing many sequence and structural similarities, CUL4B plays distinct roles in spermatogenesis from its homologous protein CUL4A. We previously reported that genetic ablation of Cul4a in mice led to male infertility because of aberrant meiotic progression. In the present study, we generated Cul4b germ cell-specific conditional knock-out (Cul4b Protein degradation via the ubiquitin-proteasome system plays a critical role during mammalian spermatogenesis. Timely removal of outlived proteins is crucial to ensure progression through different phases of spermatogenesis including mitotic, meiotic divisions, and postmeiotic morphogenesis. The ubiquitin-proteasome system selects its targets via a group of E3 ubiquitin ligases, which upon interaction with E2 ubiquitin-conjugating enzymes tag substrates for proteasomal degradation by the 26S proteasome. The largest mammalian E3 ligase family is the CRL (cullin-RING finger ubiquitin ligase) family, which includes eight members. Each cullin family member serves as a molecular scaffold and forms an E3 ligase complex with substrate-recruiting receptors and a linker protein (1). The Cul4b gene, located on the X chromosome, shares extensive sequence homology and functional redundancy with the other CRL4 family member, Cul4a. Both proteins employ DDB1 (DNA damage-binding protein 1) as the linker protein, which in turn recruits common or distinct DDB1-CUL4 associated factors for substrate binding. The DDB1-CUL4-mediated protein modification/degradation is involved in critical cellular processes including DNA replication, DNA repair, cell cycle control, and histone modifications (2).Male infertility accounts for ϳ40 -50% of infertility cases in humans (3, 4). Many factors contribute to male infertility; however, the vast majority of male infertility cases are associated with oligozoospermia (decreased sperm number), asthenozoospermia (reduced sperm motility), and/or teratozoospermia (abnormal sperm morphology). In the mammalian testis, a single pluripotent spermatogonial stem cell (SSC) 2 undergoes several rounds of mitotic divisions followed by meiotic divisions and complex postmeiotic morphogenesis, eventually giving rise to a cohort of mature spermatozoa. We have previously reported that the CRL4 proteins exhibited complementary expression patterns in adult mouse testis, where CUL4A was predominantly detected in primary spermatocytes, whereas CUL4B was highly expressed in Sertoli cells, spermatogonia, and spermatids (5). The absence of CUL4B in spermatocytes is likely due to meiotic sex chromosome inactivation, a transient X chromosome inactivation caused by sex chromosome condensation and subsequent gene silencing (6). However, the

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“…Multiple HOX proteins have been reported to be post-translationally ubiquitinated by the CUL4 ubiquitin ligase promoting their proteasome-dependent degradation [21,22]. Similarly, HOXC10 might be regulated by the anaphase promoting complex (APC) in an ubiquitin-dependent and proteasomal manner and its abundance was shown to oscillate during the cell cycle [23].…”

Section: Introductionmentioning

confidence: 99%

KPC2 relocalizes HOXA2 to the cytoplasm and decreases its transcriptional activity

Bridoux

Bergiers

Draime

et al. 2015

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4

Abstract: Key Points• The CUL4 ubiquitin ligases target HOXB4 for ubiquitination and degradation.• A degradation-resistant HOXB4 variant markedly enhanced ex vivo expansion of adult hematopoietic stem cells.

Cited by 37 publications

References 31 publications

CDK4/6 Inhibitor PD 0332991 Sensitizes Acute Myeloid Leukemia to Cytarabine-Mediated Cytotoxicity

CDK4/6 Inhibitor PD 0332991 Sensitizes Acute Myeloid Leukemia to Cytarabine-Mediated Cytotoxicity

Cell Autonomous and Nonautonomous Function of CUL4B in Mouse Spermatogenesis

KPC2 relocalizes HOXA2 to the cytoplasm and decreases its transcriptional activity

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