Differential regulation of 11β-hydroxysteroid dehydrogenase-1 by dexamethasone in glucocorticoid-sensitive and -resistant childhood lymphoblastic leukemia

Glucocorticoid therapy forms a crucial first-line treatment for childhood acute lymphoblastic leukemia (ALL). However, glucocorticoid resistance is a therapeutic problem with an unclear molecular mechanism. 11β-Hydroxysteroid dehydrogenase-1 (11β-HSD1) is expressed in glucocorticoid target tissue, where it regenerates active glucocorticoids from inert 11keto-glucocorticoids, amplifying intracellular glucocorticoid levels. Here, we show 11β-HSD1 expression in leukemic cells from ALL patients (n = 14). 11β-HSD1 was differentially regulated by glucocorticoids between glucocorticoid-sensitive and -resistant ALL cells. Dexamethasone increased 11β-HSD1 mRNA levels in glucocorticoid-sensitive ALL cells, but decreased levels in the resistant group. Our data suggest that differential induction of 11β-HSD1 contributes to the glucocorticoid sensitivity in leukemia.     

FcγRIIIa receptor interacts with androgen receptor and PIP5K1α to promote growth and metastasis of prostate cancer

Low‐affinity immunoglobulin gamma Fc region receptor III‐A (FcγRIIIa) is a cell surface protein that belongs to a family of Fc receptors that facilitate the protective function of the immune system against pathogens. However, the role of FcγRIIIa in prostate cancer (PCa) progression remained unknown. In this study, we found that FcγRIIIa expression was present in PCa cells and its level was significantly higher in metastatic lesions than in primary tumors from the PCa cohort (P = 0.006). PCa patients with an elevated level of FcγRIIIa expression had poorer biochemical recurrence (BCR)‐free survival compared with those with lower FcγRIIIa expression, suggesting that FcγRIIIa is of clinical importance in PCa. We demonstrated that overexpression of FcγRIIIa increased the proliferative ability of PCa cell line C4‐2 cells, which was accompanied by the upregulation of androgen receptor (AR) and phosphatidylinositol‐4‐phosphate 5‐kinase alpha (PIP5Kα), which are the key players in controlling PCa progression. Conversely, targeted inhibition of FcγRIIIa via siRNA‐mediated knockdown or using its inhibitory antibody suppressed growth of xenograft PC‐3 and PC‐3M prostate tumors and reduced distant metastasis in xenograft mouse models. We further showed that elevated expression of AR enhanced FcγRIIIa expression, whereas inhibition of AR activity using enzalutamide led to a significant downregulation of FcγRIIIa protein expression. Similarly, inhibition of PIP5K1α decreased FcγRIIIa expression in PCa cells. FcγRIIIa physically interacted with PIP5K1α and AR via formation of protein–protein complexes, suggesting that FcγRIIIa is functionally associated with AR and PIP5K1α in PCa cells. Our study identified FcγRIIIa as an important factor in promoting PCa growth and invasion. Further, the elevated activation of FcγRIII and AR and PIP5K1α pathways may cooperatively promote PCa growth and invasion. Thus, FcγRIIIa may serve as a potential new target for improved treatment of metastatic and castration‐resistant PCa.     

Cell Density-Dependent Proliferative Effects of Transforming Growth Factor (TGF)-β1, β2, and β3 in Human Chondrosarcoma Cells HCS-2/8 Are Associated with Changes in the Expression of TGF-β Receptor Type I

In this study, the growth properties of the human chondrosarcoma cell line HCS- 2/8, its response to transforming growth factor (TGF)-β isoforms 1, 2, and 3, and its expression of TGF-β receptors I and II were examined. We demonstrated that these tumor cells are not contact-inhibited and that they can proliferate in the absence of additional serum growth factors. In sparse cultures, all TGF-β forms inhibited the growth of HCS-2/8 cells, whereas they induced a 2-fold increase of DNA synthesis in serum-fed confluent cultures. In serum-free confluent conditions only TGF-β1 stimulated the proliferation rate, whereas TGF-β2 was without effect and TGF-β3 was rather inhibitory. This bimodal effect of TGF-β forms was associated with a greater level of TGF-β receptor I mRNA in confluent HCS-2/8 than in sparse cultures, suggesting that the growth response to TGF-β forms is dependent on the receptor profile expressed.     

Functional CD32 Molecules on Human NK Cells

Human NK cells are large granular lymphocytes that kill neoplastic or virally infected targets using perforin-dependent mechanisms. CD16 or FcγRIII is one of the cell surface molecules that can trigger the killing machinery following binding of the Fc portion of IgG to the receptor: a mechanism known as antibody dependent cell-mediated cytotoxicity (ADCC). We have recently shown that some individuals express an additional FcγR on their NK cells, CD32 or FcγRII. This receptor has now been characterized at the molecular, biochemical and functional level. The present review outlines our findings to date on the features of this novel receptor. These findings suggest that the presence of a functional FcγRII on the surface of NK cells could have important clinical consequences in both tumor immunotherapy and autoimmune disease.     

Tumor Necrosis Factor-β and Hypercalcemia

Hypercalcemia in hematological malignancy is frequently encountered in lymphoid malignancies such as adult T-cell leukemia (ATL) and multiple myeloma and is difficult to manage. As a causative agent of hypercalcemia in ATL, tumor necrosis factor-β (TNF-β), previously known as lymphotoxin, has been carefully studied and reviewed here. Bone resorption studies showed the presence of activity in culture supernatants of HTLV-I infected cells. Enzyme linked immunosorbent assays (ELISA) for TNF-β detected elevated TNF-β in the sera of ATL patients with hypercalcemia. Immunostaining by monoclonal anti-TNF-β antibody demonstrated the presence of TNF-β in both HTLV-I infected cell lines and freshly isolated ATL cells. Furthermore biological TNF-β activity assay including inhibition of anti-TNF-β antibody confirmed the conventional documentation of TNF-β activity in the sera and culture supernatants of HTLV-I infected cell lines. These studies showed that the TNF-β secreted from ATL cells might be one of the factors contributing to the hypercalcemia in patients with ATL functioning as an osteoclast activating factor (OAF).     

Hematopoietic Progenitors and Synergism of Interferon-γ and Stem Cell Factor

Interferon-γ (IFN-γ), an immunoregulatory cytokine produced by activated T cells and natural killer cells in response to viral infection or other stimuli, is generally recognized as a suppressor of hematopoiesis. IFN-γ inhibited in vitro colony formation by granulocyte-macrophage (GM), erythroid and multipotential progenitors. This cytokine exerted direct suppression on the proliferation process, but not on the commitment, of GM progenitors. The antiproliferative effects of IFN-γ may, in part, result from the prolongation of the doubling time of GM progenitors. Clinically, IFN-γ may play an important role in the pathogenesis of pancytopenia in aplastic anemia and in the hemophagocytic syndrome. However, as well as showing inhibitory effects, IFN-γ increased the number of pure and mixed megakaryocyte colonies formed by post-5-fluorouracil treated bone marrow cells and, moreover, the addition of IFN-γ to culture containing stem cell factor resulted in a synergistic effect on the development of both primitive hematopoietic progenitors and mature populations. These findings suggest that IFN-γ has bifunctional activity in hematopoiesis.     

MicroRNA‐125b suppresses the development of bladder cancer by targeting E2F3

Increasing evidence has suggested that dysregulation of certain microRNAs (miRNAs) may contribute to tumorigenesis. microRNA‐125b (miR‐125b) was implicated to have close relationship with cell proliferation and differentiation, and downregulation of miR‐125b was observed in various types of cancers. However, the biological function of miR‐125b in bladder tumorigenesis is still unknown. In our study, we showed that the expression of miR‐125b was significantly decreased in bladder cancer tissues and four bladder cancer cell lines. Moreover, miR‐125b could suppress bladder cancer cells to form colonies in vitro and to develop tumors in nude mice. E2F3, which was critical for G1/S transition and was overexpressed in most of poor‐differentiated bladder cancers, was identified as a target of miR‐125b by luciferase assay. The E2F3 mRNA and protein expression levels were detected in bladder cancer tissues and cell lines, and interestingly, inverse correlations between miR‐125b and E2F3 protein level were found in bladder cancer tissues and four E2F3 nonamplified cell lines. Introduction of miR‐125b could reduce the expression of E2F3 protein but not the E2F3 mRNA. In addition, we observed that transfection of miR‐125b could inhibit the expression of Cyclin A2, one of the E2Fs‐responsive genes involved in G1/S transition. These results suggest that miR‐125b may regulate G1/S transition through the E2F3–Cyclin A2 signaling pathway. Taken together, miR‐125b may act as a tumor suppressor in bladder urothelium, and downregulation of miR‐125b may contribute to the tumorigenesis of bladder cancer.     

The Fc‐alpha receptor is a new target antigen for immunotherapy of myeloid leukemia

Antibody‐based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64‐targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcαRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti‐human CD89 single‐chain Fragment variable and the well‐characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC₅₀ values in range 0.2–3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose‐dependent apoptosis‐mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89‐specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89‐ETA' as a potent, novel immunotherapeutic agent.     

MUC1-Tn-targeting chimeric antigen receptor-modified Vγ9Vδ2 T cells with enhanced antigen-specific anti-tumor activity

Chimeric antigen receptor (CAR) αβ T cell adoptive immunotherapy has shown great promise for improving cancer treatment. However, there are several hurdles to overcome for the wide clinical application of CAR-αβ T cells therapy, including side effects and a limited T cells source from cancer patients. Therefore, we sought to identify an alternative T cell subset that could avoid these limitations and improve the effectiveness of CAR-T immunotherapy. γδ T cells are a minor subset of T cells, which share the characteristic of innate immune cells and adaptive immune cells. Vγ9Vδ2 T cells are a predominant γδ T subset in the circulating peripheral blood. In this study, we investigated the antigen-specific antitumor activity of CAR-Vγ9Vδ2 T cells targeting MUC1-Tn antigen. Vγ9Vδ2 T cells were expanded from peripheral blood mononuclear cells of healthy volunteers with zoledronic acid and interleukin-2. CAR-Vγ9Vδ2 T cells were generated by transfection of lentivirus encoding MUC1-Tn CAR. Cytotoxicity assays with various cancer cell lines revealed that CAR-Vγ9Vδ2 T cells could effectively lyse tumor cells in an antigen-specific manner, with similar or stronger effects than CAR-αβ T cells. However, CAR-Vγ9Vδ2 T cells had shorter persistence, which could be improved with the addition of IL-2 to maintain the function of CAR-Vγ9Vδ2 T cells with consecutive stimulation of tumor cells. Using a xenograft mouse model, we further showed that CAR-Vγ9Vδ2 T cells more effectively suppressed tumor growth in vivo than Vγ9Vδ2 T cells. Therefore, MUC1-Tn CAR-modified Vγ9Vδ2 T cells may represent a novel, promising ready-to-use product for cancer allogeneic immunotherapy.     

Phenotypic characterization and prognostic impact of circulating γδ and αβ T‐cells in metastatic malignant melanoma

Human T cells carrying γδ T‐cell receptors (TCRs) represent a minor population relative to those with αβ TCRs. There has been much interest recently in the possibility of using these γδ T‐cells in cancer therapy because they can kill tumor cells in vitro in an MHC‐unrestricted manner, and possess potential regulatory capability and antigen‐presenting capacity. The presence of γδ T‐cells in late‐stage melanoma patients and their relationship with survival has not been extensively explored, although relatively lower percentages of total γδ T‐cells and Vδ2+ cells have been reported. Here, we present a detailed analysis of associations of γδ T‐cell subsets and differentiation stages with survival in Stage IV patients, compared with CD4+ and CD8+ αβ T‐cells. We found an increased Vδ1:Vδ2‐ratio and a decreased CD4:CD8‐ratio in patients compared to healthy controls, on the basis both of relative frequencies and absolute cell counts per μL blood. Nonetheless, Kaplan–Meier analyses showed that a higher than median frequency of Vδ1+ cells was negatively associated with survival, whereas there were no positive or negative associations with frequencies of Vδ2+ cells. Correlations of cell differentiation status with survival revealed a negative association of early‐differentiated Vδ1+ T cells with survival, both on the basis of relative frequencies and absolute counts. There was also a positive correlation between the frequencies of early‐differentiated CD8+ αβ T‐cells and survival. Our findings suggest peripheral blood frequencies of Vδ1+ T‐cells as a potential prognostic marker in melanoma. The mechanisms by which higher abundance of Vδ1+ cells are associated with poorer survival require determination.     

CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

Cadherin 6 (CDH6) is significantly overexpressed in advanced ovarian and renal cancers. However, the role of CDH6 in cancer metastasis is largely unclear. Here, we investigated the impact of CDH6 expression on integrin‐mediated metastatic progression. CDH6 preferentially bound to αIIbβ3 integrin, a platelet receptor scarcely expressed in cancer cells, and this interaction was mediated through the cadherin Arginine–glycine–aspartic acid (RGD) motif. Furthermore, CDH6 and CDH17 were found to interact with α2β1 in αIIbβ3^low cells. Transient silencing of CDH6, ITGA2B, or ITGB3 genes caused a significant loss of proliferation, adhesion, invasion, and lung colonization through the downregulation of SRC, FAK, AKT, and ERK signaling. In ovarian and renal cancer cells, integrin αIIbβ3 activation appears to be a prerequisite for proper α2β1 activation. Interaction of αIIbβ3 with CDH6, and subsequent αIIbβ3 activation, promoted activation of α2β1 and cell adhesion in ovarian and renal cancer cells. Additionally, monoclonal antibodies specific to the cadherin RGD motif and clinically approved αIIbβ3 inhibitors could block pro‐metastatic activity in ovarian and renal tumors. In summary, the interaction between CDH6 and αIIbβ3 regulates α2β1‐mediated adhesion and invasion of ovarian and renal cancer metastatic cells and constitutes a therapeutic target of broad potential for treating metastatic progression.     

Novel T-cell receptor δ gene rearrangement involving a recombining element located 2.6 kb 3′ from the Vδ2 gene segment

In this study, we describe a novel T-cell receptor δ (TCRδ) gene rearrangement observed in acute myeloid leukemia with coexpression of T-lymphoid antigens (Ly+AML) and in peripheral blood leukocytes (PBL) from one out of ten healthy donors. The rearrangement was identified by Southern blot analysis using a joining region (Jδ1) specific probe and amplified by polymerase chain reaction (PCR) with a variable region (Vδ2) and Jδ1 specific primers. The nucleotide sequence analysis of an atypical 3000 bp PCR product allowed localization of the breakpoint within the TCRδ gene locus, 2.6 kb 3′ from the Vδ2 gene segment. A regular Dδ2–Dδ3–Jδ1 joining was found at the 3′ end of the breakpoint, indicating that the rearrangement was mediated by the VDJ recombinase, but no TCRδ gene segment was detected at the 5′ end. Analysis of the germline sequence 3′ from the breakpoint revealed an isolated recombination signal sequence (RSS) capable of initiating a rearrangement. The RSS motif described by us is the second TCRδ recombining element (δRec2). The δRec2(Dδ)Jδ1 recombination is a rather rare event and can be found in acute leukemia and in PBL from healthy individuals. Most likely, the nonfunctional δRec2(Dδ)Jδ1 rearrangement is a transient step during the VDJ recombination. It may potentially lead to deletion of the δRec2(Dδ)Jδ1 complex and either to direct joining of a Vδ region to one of the downstream Jδ regions or to a rearrangement of the TCR gene.     

Expression of retinoic acid α and β receptor genes in liver and hepatocellular carcinoma

cDNA probes for human retinoic acid receptors α and β (RARα and RARβ) were modified for use as specific hybridization probes to study hepatocellular carcinomas (HCC) and cell lines, liver regeneration, and fetal development. RARβ mRNA was detected at low levels in adult liver and rose markedly during the early phase of liver regeneration. RARβ mRNA was present at very low levels in HCC and was not detected in fetal liver. In contrast, RARα mRNA was present at low levels in normal liver, but showed a marked elevation in several HCCs and cell lines. Growth of cell lines was altered by retinoic acid (RA), but the effects could not be predicted by the levels of either RARα or RARβ mRNA. However, the response correlated with cell phenotype. Three cell lines with an adult phenotype (high albumin and low α-fetoprotein) were inhibited by RA, two undifferentiated lines showed moderate growth stimulation, and two of three cell lines that had high levels of α-fetoprotein were markedly stimulated by RA.     

真核起始因子3b在乳腺癌中的表达及与患者临床特征的关系

目的探讨真核起始因子3b(eIF3b)在乳腺癌中的表达及与患者临床特征的关系。方法取45例乳腺癌患者的乳腺癌组织和相应的癌旁组织,采用实时定量聚合酶链反应(RT-PCR)检测乳腺癌组织和相应的癌旁组织eIF3b mRNA的相对表达量,采用免疫组化二步法检测乳腺癌组织和相应的癌旁组织e IF3b蛋白的阳性表达率,分析e IF3b mRNA的相对表达量和eIF3b蛋白的阳性表达率与乳腺癌患者临床特征的关系。结果乳腺癌组织中eIF3b m RNA的相对表达量和eIF3b蛋白的阳性表达率,均明显高于癌旁组织,差异均有统计学意义(P﹤0.01)。不同年龄、病理类型、肿瘤直径、组织学分级乳腺癌患者乳腺癌组织中eIF3b mRNA相对表达量和e IF3b蛋白阳性表达率的比较,差异均无统计学意义(P﹥0.05);不同腋窝淋巴结转移情况、TNM分期乳腺癌患者乳腺癌组织中eIF3b mRNA相对表达量和eIF3b蛋白阳性表达率的比较,差异均有统计学意义(P﹤0.05)。结论 eIF3b表达增高不仅可能参与了乳腺癌发生过程,也可能在乳腺癌的增殖、转移和侵袭过程中发挥促进作用。     

Radiobiologic significance of apoptosis and micronucleation in quiescent cells within solid tumors following γ-ray irradiation

Purpose: To determine the frequency of apoptosis in quiescent (Q) cells within solid tumors following γ-ray irradiation, using four different tumor cell lines. In addition, to assess the significance of detecting apoptosis in these cell lines.

Methods and Materials: C3H/He mice bearing SCC VII or FM3A tumors, Balb/c mice bearing EMT6/KU tumors, and C57BL mice bearing EL4 tumors received 5-bromo-2′-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received γ-ray irradiation at a dose of 4–25 Gy while alive or after tumor clamping. Immediately after irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 hours after irradiation, tumor cell suspensions obtained in the same manner were fixed. The apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU.

Results: In total cells, SCC VII, FM3A, and EMT6/KU cells showed reasonable relationships between MN frequency and surviving fraction (SF). However, fewer micronuclei were induced in EL4 cells than the other cell lines. In contrast, a comparatively close relationship between apoptosis frequency and SF was found in total cells of EL4 cell line. Less apoptosis was observed in the other cell lines. Quiescent tumor cells exhibited significantly lower values of MN and apoptosis frequency probably due to their large hypoxic fraction, similar to total tumor cells on clamped irradiation.

Conclusion: γ-ray irradiation induced MN formation in SCC VII, FM3A, and EMT6/KU tumor cells, and the apoptosis was marked in EL4 cells compared with the other cell lines. Our method for detecting the Q cell response to γ-ray irradiation using P cell labeling with BrdU and the MN frequency assay was also applicable to apoptosis detection assay.     

α-Adrenergic receptors may contribute to the hypertriglyceridemia associated with tumour growth

The inoculation of the Yoshida AH-130 ascites hepatoma to rats resulted in an important loss of adipose tissue associated with a decrease in lipoprotein lipase (LPL) activity. Tumour burden also resulted in an important hyperlipidemia which affected both triglyceride and free fatty acids. Administration of phentolamine (an α-adrenergic antagonist) to tumour-bearing rats did not influence LPL activity, but it reversed the increase in plasma triglycerides associated with tumour burden. It is suggested that the hypertriglyceridemia associated with tumour growth may be, in part, a consequence of the effect of catecholamines on hepatic triglyceride secretion, via α-adrenergic receptors.     

LncRNA MEG3 inhibits retinoblastoma invasion and metastasis by inducing β-catenin degradation

In our previous study, we found that low expression of LncRNA-MEG3 was closely associated with the invasion and metastasis of retinoblastomas. The molecular mechanism by which MEG3 inactivation induces the invasion and metastasis of retinoblastoma cell lines remains unclear. We used the GEO database to analyze the expression of MEG3 in retinoblastoma tissues and MEG3-related pathways. The scratch, transwell migration, mouse tumor metastasis, and mouse fluorescence live imaging assays were performed to detect migration and invasion of retinoblastoma cell lines. The RNA pull down, electrophoretic mobility shift, RIP, co-immunoprecipitation, and ubiquitination assays were performed to analyze the molecular mechanisms. The GEO database showed that the expression of MEG3 was low in retinoblastoma tissues and was closely associated with the invasion of retinoblastoma cells and activity of the Wnt pathway. Both in vivo and in vitro experiments confirmed that MEG3 inhibited the migration and invasion of retinoblastoma cells. Cell experiments confirmed that MEG3 could promote the binding of β-catenin and GSK-3β and induce phosphorylation, ubiquitination and degradation of β-catenin indirectly. In conclusion, MEG3 can promote the degradation of β-catenin via GSK-3β, which in turn inactivates the Wnt pathway and ultimately inhibits the invasion and metastasis of retinoblastoma cells.