Protein Kinase Regulatory Subunit 1B Research Articles

A novel human multiple myeloma cell line with a 1q21 gain genetic abnormality and CKS1B overexpression.

Multiple myeloma (MM) is an incurable hematologic malignancy mainly due to its cytogenetic abnormalities. Therefore, it is important to establish permanent malignant MM cell lines as tools to develop more effective therapies. Pleural effusion cells of a 70-year-old patient was collected to establish the CZ2 cell line. Characterization of CZ2 was determined with nephelometry, flow cytometry, fluorescence in situ hybridization (FISH), and chromosomal microarray analysis (CMA). Western blotting analysis was adopted to determine protein expression. Cell viability was measured by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We established and characterized a new MM cell line, CZ2. Using nephelometry and flow cytometry, cells with typical plasma cell morphology but not classical plasma cell phenotype were found to be non-immunoglobulin-secretary cells. FISH analysis of cells revealed a unique characteristic, namely, that there was only gain of the 1q21 region (1q21+). No other common cytogenetic abnormalities in MM, such as deletion of 17p (17p-), deletion of 13q (13q-), or translocation of immunoglobulin heavy chain (IgH), were observed. In addition, the original cell line maintains its single cytogenetic abnormality. Meanwhile, we observed through western blotting that CDC28 protein kinase regulatory subunit 1B (CKS1B), an adverse prognostic gene located in the 1q21 region, was highly expressed in CZ2. Knockdown of CKS1B reduced cell viability and also increased the levels of cleaved-poly(ADP-ribose) polymerase (cleaved-PARP) and cleaved-caspase3. CZ2 provides a suitable material for cellular and molecular studies of MM with only a 1q21 abnormality. This cell line is characterized by a gain of 1q21, and the high expression of CKS1B is an important model for studies of myeloma cell growth and drug resistance during therapy.

Regulation of microRNA miR-197-3p/CDC28 protein kinase regulatory subunit 1B (CKS1B) axis by Circular RNA hsa_circ_0000285 promotes glioma progression

ABSTRACT Circular RNA circ_0000285 is differentially expressed in several malignancies; however, its role in gliomas is under investigation. Reverse transcription quantitative polymerase chain reaction was conducted to evaluate the expression of circ_0000285, miR-197-3p, and CDC28 protein kinase regulatory subunit 1B (CKS1B) in glioma tissues and cells. Cell Counting Kit-8 and Transwell invasion assays coupled with Western blotting analysis using anti-Bax and anti-Bcl-2 antibodies were performed to evaluate cell proliferation, invasion, and apoptosis. Luciferase reporter and AGO2 RNA immunoprecipitation assays were conducted to verify the interaction between miR-197-3p and circ_0000285 or CKS1B. Xenograft tumor growth was evaluated in mice. We noted that circ_0000285 was highly expressed in glioma tissues and cells and that circ_0000285-silencing retarded tumor growth both in vitro and in vivo. This effect was mediated by the binding of circ_0000285 to miR-197-3p, which silenced CKS1B, an essential driver of glioma cell proliferation and invasion. Thus, circ_0000285 boosted glioma progression by regulating the miR-197-3p/CKS1B axis, highlighting a novel competing endogenous RNA circuit of glioma progression.

The Clinical Significance and Potential Molecular Mechanism of Upregulated CDC28 Protein Kinase Regulatory Subunit 1B in Osteosarcoma.

Background CDC28 Protein Kinase Regulatory Subunit 1B (CKS1B) is a member of cyclin-dependent kinase subfamily and the relationship between CKS1B and osteosarcoma (OS) remains to be explored. Methods 80 OS and 41 nontumor tissue samples were arranged to conduct immunohistochemistry (IHC) to evaluate CKS1B expression between OS and nontumor samples. The standard mean deviation (SMD) was calculated based on in-house IHC and tissue microarrays and exterior high-throughput datasets for further verification of CKS1B expression in OS. The effect of CKS1B expression on clinicopathological and overall survival of OS patients was measured through public high-throughput datasets, and analysis of immune infiltration and single-cell RNA-seq was applied to ascertain molecular mechanism of CKS1B in OS. Results A total of 197 OS samples and 83 nontumor samples (including tissue and cell line) were obtained from in-house IHC, microarrays, and exterior high-throughput datasets. The analysis of integrated expression status demonstrated upregulation of CKS1B in OS (SMD = 1.38, 95% CI [0.52–2.25]) and the significant power of CKS1B expression in distinguishing OS samples from nontumor samples (Area under the Curve (AUC) = 0.89, 95% CI [0.86–0.91]). Clinicopathological and prognosis analysis indicated no remarkable significance but inference of immune infiltration and single-cell RNA-seq prompted that OS patients with overexpressed CKS1B were more likely to suffer OS metastasis while MYC Protooncogene may be the upstream regulon of CKS1B in proliferating osteoblastic OS cells. Conclusions In this study, sufficient evidence was provided for upregulation of CKS1B in OS. The advanced effect of CKS1B on OS progression indicates a foreground of CKS1B as a biomarker for OS.

In Multiple Myeloma, High-Risk Secondary Genetic Events Observed at Relapse Are Present from the Diagnosis in Tiny Undetectable Subclones

In Multiple Myeloma, High-Risk Secondary Genetic Events Observed at Relapse Are Present from the Diagnosis in Tiny Undetectable Subclones

Super-Enhancer Driven Regulation of CKS1B in Multiple Myeloma: Implications in Mediating Response to BET Inhibitor and Celmod Agent Combination

Introduction: The Myeloma Genome Project (MGP) characterized the genomic landscape of patients with newly diagnosed multiple myeloma (NDMM) (Walker BA, et al. Blood 2018; 132[6]:587-597). Using a multi-omics unsupervised clustering approach, 12 molecularly-defined disease segments were identified (Ortiz M, et al. Blood 2018; 132[suppl 1]:3165). Here, we performed experimental validation of CDC28 Protein Kinase Regulatory Subunit 1B (CSK1B) that was identified as a putative target from the disease segment with poorest clinical outcome. CKS1B was selected for in-depth validation due to their role in cell cycle pathways associated with high-risk disease, biological mechanisms of chromosome 1q amplification and druggability.Methods: Association of CKS1B with outcomes was analyzed in NDMM patients, across relapses and with clinical outcome datasets from MGP and Mayo clinic. Inducible shRNAs of CKS1B and bromodomain containing protein 4 (BRD4, a member of the BET [bromodomain and extra terminal domain] family) were generated in MM cell lines. BRD4 and Aiolos ChIP-seq datasets were analyzed for binding on CKS1B gene. BRD4 inhibitors JQ1 and CC-90010 were utilized for inhibition studies in MM cell lines.Results: Higher expression of CKS1B was associated with significantly poorer PFS, OS, disease severity and relapse. Knock-down of CKS1B in MM cells led to a significant decrease in proliferation (P<0.001) and enhanced apoptosis in MM cell lines. BRD4-ChIP sequencing studies revealed that the expression of CKS1B was regulated by super-enhancer (SE) associated elements. As expected, two BRD4 inhibitors, JQ1 and CC-90010 and inducible BRD4 shRNAs downregulated the expression of CKS1B resulting in decreased proliferation, cell cycle arrest and apoptosis in MM cell lines. Furthermore, MM cell lines harboring chromosome 1q gain/amp showed higher sensitivity to BRD4 inhibition compared to cell lines with normal 1q copy number. Mechanistic studies revealed that BRD4inh and BRD4 shRNAs downregulated the expression of Aiolos and Ikaros in MM cell lines. Interestingly, Aiolos ChIP-sequencing studies demonstrated the binding of Aiolos at the transcriptional start sites of CKS1B with the transcriptional activation mark. The immunomodulatory agent (IMiD ®) pomalidomide (Pom) transcriptionally downregulated CKS1B in Pom-sensitive cells downstream of Aiolos, Ikaros degradation. Based on these mechanisms, IMiD agents, lenalidomide, Pom and the novel Cereblon E3 ligase modulating degrader (CELMoD ®) agent CC-92480 in combination with BRD4inh promoted a synergistic decrease in proliferation, cell cycle arrest and increase in apoptosis in both Pom-sensitive and -resistant cell lines. The combination of IMiD or novel CELMoD agent with BRD4inh also promoted deeper downregulation of CKS1B, Aiolos, Ikaros, c-Myc and survivin proteins with enhanced levels of apoptotic marker cleaved Caspase 3 as compared to single agents alone.Conclusions: In summary, we have identified CKS1B as a key target associated with poor outcome in MM patients. Translational studies suggest a profound downregulation of CKS1B and key pro-survival effector proteins following combination treatment with BRD4inh and IMiD agents/novel CELMoD agents resulting in synergistic anti-tumor effects. These data provide rationale for testing these agents in the clinic for high-risk and IMiD-relapsed patients.Figure: Changes in cell proliferation and protein levels of key signaling mediators were studied in K12PE cell line treated with increasing doses of Lenalidomide, Pomalidomide and CC-92480 in combination with JQ1. [Display omitted] DisclosuresAhsan: BMS: Current Employment, Current equity holder in publicly-traded company. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hsu: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bahlis: Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Molecular Network Profiling in Intestinal- and Diffuse-Type Gastric Cancer.

Simple SummaryCancer has several phenotypic subtypes where the responsiveness towards drugs or capacity of migration or recurrence are different. The molecular networks are dynamically altered in various phenotypes of cancer. To reveal the network pathways in epithelial-mesenchymal transition (EMT), we have profiled gene expression in mesenchymal stem cells and diffuse-type gastric cancer (GC), as well as intestinal-type GC. Gene expression signatures revealed that the molecular pathway networks were altered in intestinal- and diffuse-type GC. The artificial intelligence (AI) recognized the differences in molecular network pictures of intestinal- and diffuse-type GC.Epithelial-mesenchymal transition (EMT) plays an important role in the acquisition of cancer stem cell (CSC) feature and drug resistance, which are the main hallmarks of cancer malignancy. Although previous findings have shown that several signaling pathways are activated in cancer progression, the precise mechanism of signaling pathways in EMT and CSCs are not fully understood. In this study, we focused on the intestinal and diffuse-type gastric cancer (GC) and analyzed the gene expression of public RNAseq data to understand the molecular pathway regulation in different subtypes of gastric cancer. Network pathway analysis was performed by Ingenuity Pathway Analysis (IPA). A total of 2815 probe set IDs were significantly different between intestinal- and diffuse-type GC data in cBioPortal Cancer Genomics. Our analysis uncovered 10 genes including male-specific lethal 3 homolog (Drosophila) pseudogene 1 (MSL3P1), CDC28 protein kinase regulatory subunit 1B (CKS1B), DEAD-box helicase 27 (DDX27), golgi to ER traffic protein 4 (GET4), chromosome segregation 1 like (CSE1L), translocase of outer mitochondrial membrane 34 (TOMM34), YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), ribonucleic acid export 1 (RAE1), par-6 family cell polarity regulator beta (PARD6B), and MRG domain binding protein (MRGBP), which have differences in gene expression between intestinal- and diffuse-type GC. A total of 463 direct relationships with three molecules (MYC, NTRK1, UBE2M) were found in the biomarker-filtered network generated by network pathway analysis. The networks and features in intestinal- and diffuse-type GC have been investigated and profiled in bioinformatics. Our results revealed the signaling pathway networks in intestinal- and diffuse-type GC, bringing new light for the elucidation of drug resistance mechanisms in CSCs.

Circular RNA NOX4 promotes the development of colorectal cancer via the microRNA‑485‑5p/CKS1B axis.

Colorectal cancer (CRC) is a common malignancy globally. The aim of the present study was to explore the role and the working mechanism of circular RNA NADPH oxidase 4 (circNOX4; circBase ID, hsa_circ_0023990) in CRC. Reverse transcription-quantitative (RT-q)PCR was used to examine the expression of circNOX4, NOX4 mRNA and microRNA (miR)-485-5p in CRC tissues and cell lines. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assays were performed to analyze CRC cell viability and motility. The glycolytic ability of CRC cells was assessed by measuring glucose consumption, lactate production, extracellular acidification and O2 consumption rates using commercial kits. The starBase database was used to predict the targets of circNOX4 and miR-485-5p, and the interaction was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. A murine xenograft model was established to verify the role of circNOX4 in CRC in vivo. The results demonstrated that the expression of circNOX4 was upregulated in CRC tissues and cell lines compared with that in adjacent normal tissues and a normal colon epithelial cell line, respectively. The expression of circNOX4 was negatively associated with the prognosis of patients with CRC. CircNOX4 silencing suppressed the proliferation, migration, invasion and glycolysis of CRC cells. miR-485-5p was identified as a target of circNOX4. CircNOX4 promoted CRC progression by sponging miR-485-5p. miR-485-5p was demonstrated to bind to the 3′ untranslated region (UTR) of CDC28 protein kinase regulatory subunit 1B (CKS1B). miR-485-5p overexpression-mediated malignant properties of CRC cells were partly reversed by the transfection with the CKS1B overexpression plasmid. CircNOX4 silencing restrained the CRC xenograft growth in vivo. Collectively, the results of the present study demonstrated that circNOX4 may serve an oncogenic role in CRC by promoting the proliferation, migration, invasion and glycolysis of CRC cells via the miR-485-5p/CKS1B axis.

Globular C1q Receptor (gC1qR/p32/HABP1) Suppresses the Tumor-Inhibiting Role of C1q and Promotes Tumor Proliferation in 1q21-Amplified Multiple Myeloma.

Immunodeficiencies are widely becoming known as important features of multiple myeloma (MM) and may promote the proliferation of malignant cells as well as confer resistance to therapy. Few studies focus on the immunomodulatory effects of the complement system on MM. This study aims to explore the role of C1q in MM patients. Plasma C1q was found to be significantly reduced in MM patients, and the amount of C1q deposited around the CD138+ cells in bone marrow (BM) biopsy sections was observed to be much higher, especially in the subgroup with 1q21 amplification (Amp1q21). CD138+ cells expressed higher levels of C1q receptors (C1qRs) than CD138− cells. Patients with Amp1q21 expressed higher levels of globular C1qR (gC1qR), whereas patients without Amp21 expressed higher levels of collagen tail C1qR (cC1qR). Additionally, gC1qR was noted to suppress the MM-inhibiting role of C1q in H929, U266, and MM1S. gC1qR interacts with insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which also suppressed the function of C1q and regulated CDC28 protein kinase regulatory subunit 1B (CKS1B) mRNA. In summary, gC1qR suppressed the MM-inhibiting role of C1q and regulated CKS1B mRNA in promoting tumor proliferation via IGF2BP3 in 1q21-amplified MM. Our findings provide novel evidence on how MM cells evade the immune system and promote survival as well as suggest possible novel targets for future therapies of MM.

Global Mapping of H3K4 Trimethylation (H3K4me3) and Transcriptome Analysis Reveal Genes Involved in the Response to Epidemic Diarrhea Virus Infections in Pigs.

Simple SummaryPorcine epidemic diarrhea virus seriously threatens the health of suckling pigs. In this study, global mapping of H3K4me3 and transcriptomic analyses in the jejunum of porcine epidemic diarrhea virus (PEDV)-infected and healthy piglets were performed by using chromatin immunoprecipitation sequencing and RNA-seq techniques. A subset of genes and H3K4 trimethylation (H3K4me3) histone modifications that are associated with PEDV infections were identified. The results revealed previously unknown and intriguing elements involved in the regulation of genes responsive to PEDV infections, which may aid in identifying key regulators and genetic resistant markers for PEDV infections.Porcine epidemic diarrhea virus (PEDV) is currently detected as the main pathogen causing severe diarrhea in pig farms. The phenotypic alterations induced by pathogenic infections are usually tightly linked with marked changes in epigenetic modification and gene expression. We performed global mapping of H3K4 trimethylation (H3K4me3) and transcriptomic analyses in the jejunum of PEDV-infected and healthy piglets using chromatin immunoprecipitation sequencing and RNA-seq techniques. A total of 1885 H3K4me3 peaks that are associated with 1723 genes were characterized. Moreover, 290 differentially expressed genes were identified, including 104 up-regulated and 186 down-regulated genes. Several antiviral genes including 2’-5’-oligoadenylate synthetase 1 (OAS1), 2’-5’-oligoadenylate synthetase 2 (OAS2), ephrin B2 (EFNB2), and CDC28 protein kinase regulatory subunit 1B (CKS1B) with higher H3K4me3 enrichment and expression levels in PEDV-infected samples suggested the potential roles of H3K4me3 deposition in promoting their expressions. Transcription factor annotation analysis highlighted the potential roles of two transcription factors interferon regulatory factor 8 (IRF8) and Kruppel like factor 4 (KLF4) in modulating the differential expression of genes involved in PEDV infection. The results provided novel insights into PEDV infection from the transcriptomic and epigenetic layers and revealed previously unknown and intriguing elements potentially involved in the host responses.

CKS1BP7, a Pseudogene of CKS1B, is Co-Amplified with IGF1R in Breast Cancers.

Pseudogenes have been reported to exhibit functional roles. Amplification or overexpression of CDC28 protein kinase regulatory subunit 1B (CKS1B) was found in various human cancers. But it was known little about CKS1B pseudogene 7 (CKS1BP7), a pseudogene sharing considerable sequence identity with CKS1B. The aim of this study was to evaluate copy number alterations (CNAs) of CKS1BP7 and address its potential roles in breast cancer. We detected copy numbers of CKS1BP7 and insulin-like growth factor 1 receptor (IGF1R) using quantitative multi-gene fluorescence in situ hybridization (QM-FISH) technique, compared their status in both invasive carcinoma and ductal carcinoma in situ (DCIS) components within the same tumors, and investigated the associations of CNAs with tumor features and patients outcomes. Amplification of CKS1BP7 (dot-like pattern) was found in 28.8% of all cases, while amplified IGF1R (cluster pattern) was identified in 24.2% of all patients. The two events often co-existed (p=0.01). Within the same tumors, identical CNAs of CKS1BP7 and IGF1R were found in DCIS and invasive carcinoma. Moreover, amplification of both genes was more frequent in aneuploidy tumors and the tumors with high ki67, but wasn't associated with patients' outcome. In summary, CKS1BP7 amplification is a frequent event in breast cancer and often co-occurs with amplified IGF1R, which provides evidence supporting the interactions between CKS1BP7 and IGF1R during mammary carcinogenesis. Our findings suggest that CKS1BP7 as well as IGF1R may serve as potential biomarkers for early detection and predict prognosis in breast cancer.

Loss of miR-1258 contributes to carcinogenesis and progression of liver cancer through targeting CDC28 protein kinase regulatory subunit 1B.

Hepatocellular carcinoma (HCC) is the leading cause of cancer related death worldwide. The number of deaths is proportional to the global incidence, which highlights the aggressive tumor biology and lack of effective therapies. Dysregulation of microRNAs has been implicated in carcinogenesis and progression of liver cancer. Here, we identified that miR-1258 was significantly downregulated in HCC and associated with poor patients' survival. Overexpression of miR-1258 significantly inhibits liver cancer cell growth, proliferation and tumorigenicity through increasing cell cycle arrest in G0/G1 phase and promotes cell apoptosis. Interestingly, stable overexpression of miR-1258 suppresses cell migration, stemness and increases sensitivity of HCC cells to chemotherapy drug like doxorubicin. The CDC28 protein kinase regulatory subunit 1B (CKS1B) was identified as a functional downstream target of miR-1258. Re-expression of CKS1B overcomes miR-1258 induced apoptosis and increases stemness of HCC cells, suggesting that loss of miR-1258 contributes to carcinogenesis and progression of liver cancer through targeting CKS1B. Therefore, loss of miR-1258 may be a potential diagnostic and prognostic biomarker and blocking miR-1258-CKS1B axis is a potential therapeutic strategy in HCC.

Overexpression of CDC28 protein kinase regulatory subunit 1B confers an independent prognostic factor in nasopharyngeal carcinoma

Data mining on public domain identified that CDC28 protein kinase regulatory subunit 1B (CKS1B) transcript was highly expressed in nasopharyngeal carcinoma (NPC). The expression of CKS1B protein and its clinicopathological associations in patients with NPC were further evaluated. Immunoexpression of CKS1B was retrospectively assessed in biopsies of 124 consecutive NPC patients without initial distant metastasis and treated with consistent guidelines. The correlations between CKS1B immunoexpression levels and clinicopathological features, as well as patient survivals, were analyzed. High CKS1B expression (49.2%) was correlated with the 7th American Joint Committee on Cancer (AJCC) stage (p = 0.014). In multivariate analyses, high CKS1B expression emerged as an independent prognostic factor for worse disease-specific survival (p < 0.001), metastasis-free survival (p < 0.001), and local recurrence-free survival (p = 0.001). High expression of CKS1B is common and associated with adverse prognostic factors and might confer tumor aggressiveness through dysregulation of the cyclin-dependent protein kinase (intrinsic regulatory activity) during cell cycle progression.

CKS1B (CDC28 protein kinase regulatory subunit 1B)

Review on CKS1B (CDC28 protein kinase regulatory subunit 1B), with data on DNA, on the protein encoded, and where the gene is implicated.

CDC28 protein kinase regulatory subunit 1B (CKS1B) expression and genetic status analysis in oral squamous cell carcinoma.

CKS1B is a member of the highly conserved cyclin kinase subunit 1 (CKS1) protein family which interacts with cyclin-dependent kinases and plays a critical role in cell cycle progression. In oral squamous cell carcinoma (OSCC), as in other malignancies, CKS1B overexpression has been correlated with reduced survival. To our knowledge, no studies evaluating the genetic status of CKS1B gene in OSCC have been reported. Herein, genetic and protein status of CKS1B were analyzed by immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) techniques in a series of primary OSCC (n=51) and lymph node OSCC metastases samples (n=14). The observed results were compared with those obtained in either inflammatory (oral lichen planus [OLP]) (n=13) and premalignant oral mucosal lesions (oral leukoplakia) (n=16). A significant CKS1B overexpression was observed in OSCC and lymph node metastases samples than in OLP and oral leukoplakia (mean 70% vs 35%, p<0.001). CKS1B overexpression correlated with p27 loss of expression (p=0.0013) and SKP2 overexpression (p<0.00). FISH study disclosed statistical differences in both gene amplifications and gains between samples corresponding to OSCC and metastases from those of OLP and leukoplakia (p<0.001). Amplifications were present in 53% of OSCC samples and 33% of lymph node metastases vs 14% of oral leukoplakia and 0% of OLP biopsy specimens (p=0.002). Polysomies of chromosome 1 were seen in 46% of OSCC, 33% of ganglionar metastases, 14% of oral leukoplakia and 10% of OLP (p=0.036). Correlation of CKS1B over-expression and gains (both polysomies and amplifications) determined by FISH was statistically significant (p<0.001). Our results indicate that a high CKS1B expression is a common finding in primary OSCC which correlates with p27 low expression and SKP2 overexpression. This phenomenon may be due either to numerical (chromosome 1 polysomy) or structural (amplifications) CKS1B genetic abnormalities. This phenotypical and cytogenetic profile is not observed in premalignant or inflammatory oral mucosal lesions.

In CD28-costimulated human naïve CD4+ T cells, I-κB kinase controls the expression of cell cycle regulatory proteins via interleukin-2-independent mechanisms.

Stimulation of naïve CD4(+) T cells through engagement of the T-cell receptor (TCR) and the CD28 co-receptor initiates cell proliferation which critically depends on interleukin (IL)-2 secretion and subsequent autocrine signalling via the IL-2 receptor. However, several studies indicate that in CD28-costimulated T cells additional IL-2-independent signals are also required for cell proliferation. In this study, using a neutralizing anti-human IL-2 antibody and two selective, structurally unrelated, cell-permeable I-κB kinase (IKK) inhibitors, BMS-345541 and PS-1145, we show that in human naïve CD4(+) T cells stimulated through a short engagement of the TCR and the CD28 co-receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin-dependent kinase 2 (CDK2) and the stability of the F-box protein S-phase kinase-associated protein 2 (SKP2) and its co-factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL-2-independent mechanisms.

Differential expression of CKS-1B in typical and blastoid variants of mantle cell lymphoma

Mantle cell lymphoma is a distinct type of B-cell lymphoma characterized by the t(11;14)(q13;q32). Mantle cell lymphomas exhibit a spectrum of morphologic findings, of which a subset of tumors is clinically aggressive with a high proliferation rate. These neoplasms are known as aggressive variants of which there are blastoid and pleomorphic subsets. CKS-1B (CDC28 protein kinase regulatory subunit 1B) is essential for the ubiquitination and degradation of p27 and cell cycle progression. We analyzed CKS-1B expression in mantle cell lymphoma cell lines and tumors by Western blot and immunohistochemical analysis. In 4 mantle cell lymphoma cell lines, CKS-1B was expressed at variable levels and correlated inversely with p27 expression. In mantle cell lymphoma tumors, CKS-1B was positive in 10 (28.6%) of 35 typical versus 14 (87.5%) of 16 blastoid/pleomorphic cases (Fisher exact test, P = .0002). Analyzed as a continuous variable, the percentage of CKS-1B-positive cells significantly correlated with blastoid/pleomorphic morphology (Mann-Whitney U test, P = .001). Twelve (23.5%) of 51 mantle cell lymphoma tumors expressed p27. Proliferation rate (Ki-67) was higher in blastoid/pleomorphic variants than in typical mantle cell lymphoma tumors and was inversely associated with p27 levels in typical mantle cell lymphoma. However, CKS-1B expression did not correlate with p27 expression, proliferation rate, or prognosis in the entire study group. Fluorescence in situ hybridization analysis of 10 CKS-1B-positive mantle cell lymphoma tumors showed no evidence of CKS-1B gene amplification. We conclude that CKS-1B is commonly expressed in mantle cell lymphoma, particularly in aggressive histologic variants, and may be involved in pathogenesis.

Interaction of Stem Cells and Their Niche: Behavior and Gene Expression Profiles of CD34+/CD38− Cells upon Co-Cultivation with AFT024.

Cell-cell contact between stem cells and cellular determinants of the microenvironment plays an essential role in the regulation of self-renewal and differentiation. The stromal cell line derived from murine fetal liver (AFT024) has been shown to support maintenance of primitive human hematopoietic progenitor cells (HPC) in vitro. We have studied the interaction between HPC (defined as CD34+/CD38− umbilical cord blood cells) and AFT024 and the impact of co-cultivation on the behavior and gene expression of HPC. By time lapse microscopy the mobility and behavior of CD34+/CD38− cells were monitored. Approximately 30% of the CD34+/CD38− cells adhered to the cellular niche through an uropod. CD44 and CD34 were co-localized at the site of contact. Gene expression profiles of CD34+/CD38− cells were then compared upon co-cultivation either with or without AFT024. After cultivation for 16h, 20h, 48h or 72h the HPC were separated form the feeder layer cells by a second FAC-Sort. Differential gene expression was analyzed using our Human Genome cDNA Microarray of over 51,145 ESTs. Among the genes with the highest up-regulation in contact with AFT024 were several genes involved in cell adhesion, proliferation and DNA-modification including tubulin genes, ezrin, complement component 1 q subcomponent 1 (C1QR1), proto-oncogene proteins c-fos and v-fos, proliferating cell nuclear antigen (PCNA), HLA-DR, gamma-glutamyl hydrolase (GGH), minichromosome maintenance deficient 6 (MCM6), uracil-DNA glycolase (UNG) and DNA-methyltransferase 1 (DNMT1). In contrast, genes that were down-regulated after contact with AFT024 included collagenase type iv (MMP2), elastin (ELN) and hemoglobin genes. Differential expression of six genes was confirmed by RT-PCR. Other authors have reported on the differential gene expression profiles of CD34+ cells derived from the bone marrow versus those from G-CSF mobilized blood. As CD34+ cells from the bone marrow might represent cells exposed to the natural HPC niche we have then compared our findings with these experiments. In these comparisons we identified several overlapping genes that are involved in regulation of cell cycle and DNA repair including PCNA, DNMT1, MCM6, MCM2, CDC28 protein kinase regulatory subunit 1B (CKS1B), Topoisomerase II (TOP2a), DNA Ligase 1 (LIG1) and DNA mismatch repair protein MLH1. All these genes were up-regulated among CD34+/CD38− cells upon co-culture with AFT024, as well as among CD34+ cells derived from the bone marrow versus those from peripheral blood. Our studies support the hypothesis that intimate contact and adhesive interaction of HPC with their niche profoundly influenced their proliferative potential and their differentiation program.