The mouse Mageb18 gene encodes a ubiquitously expressed type I MAGE protein and regulates cell proliferation and apoptosis in melanoma B16-F0 cells

Although many cancer vaccines have been developed against type?I MAGE (melanoma antigen) genes owing to their shared tumour-specific expression properties, studies about their expression and functions are relatively limited. In the present study, we first identify a non-testis-specific type?I MAGE gene, Mageb18 (melanoma antigen family B 18). Mouse Mageb18 is also expressed in digestion- and immune-related tissues as well as testis, and its expression in testis is age-dependent. Mageb18 is expressed in many mouse-derived cell lines, and DNA demethylation and histone acetylation mediate the reactivation of Mageb18 in Mageb18-negtive H22 and C6 cells. We also show that mouse Mageb18 encodes a 46?kDa protein which is predominantly localized in the cytoplasm. In testis, the endogenous MAGEB18 protein is mainly expressed in proliferative spermatogonia and primary and secondary spermatocytes, but less so in spermatids. Finally, we demonstrate that knockdown of MAGEB18 inhibits the growth of B16-F0 cells and induces apoptosis, which correlates with increased levels of TP53 (tumour protein 53), p21, Bax and caspase 3. The results of the present study thus uncover an important phenomenon that the expression of certain type?I MAGE genes, at least for Mageb18, is non-testis-specific. Although they can regulate various malignant phenotypes of cancer cells, it is necessary to study further their expression pattern in normal tissues before using them to develop more effective and safer cancer vaccines.     

Regulation of melanin synthesis by the TGF-β family in B16 melanoma cells

Effects of representative members of the transforming growth factor-β (TGF-β) family, TGF-β1, activin A and BMP-2, on melanin content and expression of pigment-producing enzymes were examined in B16 melanoma cells. Treatment with TGF-β1 or activin A but not with BMP-2 significantly decreased melanin content and expression of Tyrosinase and Tyrp-1, suggesting an inhibitory effect of TGF-β1 and activin A on melanin synthesis. TGF-β1 completely inhibited melanin synthesis induced by α-melanin stimulating hormone (α-MSH), whereas activin A only slightly did. As compared with parental B16 cells, the inhibitory effects of TGF-β1 and activin A on melanin content were relative smaller in B16 F10 cells, a subline of B16 cells that contain more pigment. The present study indicates that in addition to TGF-β, activin negatively regulates melanogenesis in the absence of α-MSH, but that the activity in the presence of α-MSH was slightly different between TGF-β and activin.     

β-Glucosidase activity in fetal bovine serum renders the plant glucoside,hypoxoside, cytotoxic toward B16-F10-BL-6 mouse melanoma cells

Summary By usingp-nitrophenyl-β-d-glucopyranoside as substrate, β-glucosidase activity was observed in fetal bovine serum (FBS). This activity could be inhibited by heat inactivation of the serum. Gel chromatography of FBS indicated the presence of β-glucosidase activity with an apparent molecular mass of 29 kDa. In McCoy’s 5A medium supplemented with non-heat inactivated FBS, the diglucoside hypoxoside ([E]-1,5-bis[4′β-d-glucopyranosyloxy-3′-hydroxyphenyl]pent-4-en-1-yne) showed cytotoxicity toward B16-F10-BL-6 mouse melanoma cells. In incubations where the media were supplemented with FBS previously heat inactivated at 56° C for 1 h or more, no cytotoxicity was observed in the presence of hypoxoside. The aglucone of hypoxoside, rooperol ([E]-1,5-bis[3′,4′-dihydroxyphenyl]pent-4-en-1-yne), showed cytotoxicity regardless of whether the serum was heat inactivated or not. The kinetics of the heat inactivation of the β-glucosidase activity in FBS coincided with the loss of apparent cytotoxicity of hypoxoside. High performance liquid chromatography analysis showed that rooperol could be generated by incubation of hypoxoside in non-heat inactivated FBS, but that this ability was lost in serum that was heat inactivated for 1 h or longer. Newborn bovine serum did not contain any β-glucosidase activity whereas it was found in three different commercial sources of FBS. This observation is of practical importance because conventional heat inactivation of FBS at 56° C for 30 min was not sufficient to inactivate the β-glucosidase activity completely.     

Down-regulation of Pkd2 by siRNAs suppresses cell–cell adhesion in the mouse melanoma cells

The Pkd2 gene encodes an integral protein (~130 kDa), named polycystin-2 (PC-2). PC-2 is mainly involved in autosomal dominant polycystic kidney disease. Recently, polycystin-1/polycystin-2 complex has been shown to act as an adhesion complex mediating or regulating cell–cell or cell–matrix adhesion, suggesting that PC-2 may play a role in cell–cell/cell–matrix interactions. Here, we knocked down the expression of Pkd2 gene with small interfering RNAs (siRNAs) in the mouse melanoma cells (B16 cells), indicating that the cells transfected with the targeted siRNAs significantly suppressed cell–cell adhesion, but not cell–matrix adhesion, compared to the cells transfected with non-targeted control (NC) siRNA. This study provides the first directly functional evidence that PC-2 mediates cell–cell adhesion. Furthermore, we demonstrated that PC-2 modulated cell–cell adhesion may be, at least partially, associated with E-cadherin. Collectively, these findings for the first time showed that PC-2 may mediate cell–cell adhesion, at least partially, through E-cadherin.     

Loss of Purkinje cells in the PKCγ H101Y transgenic mouse

Spinocerebellar ataxia type 14 (SCA14) is an autosomal, dominant neurodegenerative disorder caused by mutations in PKCγ. The objective of this study was to determine effects of PKCγ H101Y SCA14 mutation on Purkinje cells in the transgenic mouse. Results demonstrated that wild type PKCγ-like Purkinje cell localization of HA-tagged PKCγ H101Y mutant proteins, altered morphology and loss of Purkinje cells were observed in the PKCγ H101Y SCA14 transgenic mouse at four weeks of age. Failure of stereotypical clasping responses in the hind limbs of transgenic mice was also observed. Further, PKCγ H101Y SCA14 mutation caused lack of total cellular PKCγ enzyme activity, loss of connexin 57 phosphorylation on serines, and activation of caspase-12 in the PKCγ H101Y SCA14 transgenic mouse. Results clearly demonstrate a need for PKCγ control of gap junctions for maintenance of Purkinje cells. This is the first transgenic mouse to our knowledge which models a human SCA14 mutation.     

Activated mouse CD4+Foxp3? T cells facilitate melanoma metastasis via Qa-1-dependent suppression of NK-cell cytotoxicity

The regulatory activities of mouse CD4⁺Foxp3⁺ T cells on various immune cells, including NK cells, have been well documented. Under some conditions, conventional CD4⁺Foxp3⁻ T cells in the periphery are able to acquire inhibitory function on other T cells, but their roles in controlling innate immune cells are poorly defined. As a potential cellular therapy for cancer, ex vivo activated CD4⁺Foxp3⁻ effector T cells are often infused back in vivo to suppress tumor growth and metastasis. Whether such activated T cells could affect NK-cell control of tumorigenesis is unclear. In the present study, we found that mitogen-activated CD4⁺Foxp3⁻ T cells exhibited potent suppressor function on NK-cell proliferation and cytotoxicity in vitro, and notably facilitated B16 melanoma metastasis in vivo. Suppression of NK cells by activated CD4⁺Foxp3⁻ T cells is cell-cell contact dependent and is mediated by Qa-1:NKG2A interaction, as administration of antibodies blocking either Qa-1 or NKG2A could completely reverse this suppression, and significantly inhibited otherwise facilitated melanoma metastasis. Moreover, activated CD4⁺Foxp3⁻ cells from Qa-1 knockout mice completely lost the suppressor activity on NK cells, and failed to facilitate melanoma metastasis when transferred in vivo. Taken together, our findings indicate that innate anti-tumor response is counter regulated by the activation of adaptive immunity, a phenomenon we term as “activation-induced inhibition”. Thus, the regulatory role of activated CD4⁺Foxp3⁻ T cells in NK-cell activity must be taken into consideration in the future design of cancer therapies.     

Angiotensin II stimulates H?-ATPase activity in intercalated cells from isolated mouse connecting tubules and cortical collecting ducts

Intercalated cells in the collecting duct system express V-type H(+)-ATPases which participate in acid extrusion, bicarbonate secretion, and chloride absorption depending on the specific subtype. The activity of H(+)-ATPases is regulated by acid-base status and several hormones, including angiotensin II and aldosterone. Angiotensin II stimulates chloride absorption mediated by pendrin in type B intercalated cells and this process is energized by the activity of H(+)-ATPases. Moreover, angiotensin II stimulates bicarbonate secretion by the connecting tubule (CNT) and early cortical collecting duct (CCD). In the present study we examined the effect of angiotensin II (10 nM) on H(+)-ATPase activity and localization in isolated mouse connecting tubules and cortical collecting ducts. Angiotensin II stimulated Na(+)-independent intracellular pH recovery about 2-3 fold, and this was abolished by the specific H(+)-ATPase inhibitor concanamycin. The effect of angiotensin II was mediated through type 1 angiotensin II receptors (AT(1)-receptors) because it could be blocked by saralasin. Stimulation of H(+)-ATPase activity required an intact microtubular network--it was completely inhibited by colchicine. Immunocytochemistry of isolated CNT/CCDs incubated in vitro with angiotensin II suggests enhanced membrane associated staining of H(+)-ATPases in pendrin expressing intercalated cells. In summary, angiotensin II stimulates H(+)-ATPases in CNT/CCD intercalated cells, and may contribute to the regulation of chloride absorption and bicarbonate secretion in this nephron segment.     

Developmental changes in regulation of the Na+, K+-ATPase α3 isoform by thyroid hormone in ferret heart

Ferret heart expresses the α1- as well as the α3-isoform of the Na⁺, K⁺-ATPase. We have shown previously that the α3 isoform is differentially upregulated during postnatal cardiac development and that in adult ferrets expression of α3 is not responsive to regulation by thyroid hormone (TH). Since developmental-stage dependent effects of TH have been reported previously, the present study examined whether effects of TH on expression of the Na⁺, K⁺-ATPase isoforms in ferret heart is modulated during development and possible mechanisms were examined. Ferrets of different age groups were treated with TH and the relative abundance of Na⁺, K⁺-ATPase isoforms in ferret myocardium was determined by immunoblotting. Thyroid hormone (T3; 50 μg/100 g body weight on 3 alternating days, s.c.) increased protein levels of the α3 isoform, but not that of α1 or β1, in myocardium of 5-day-old and 3-week-old ferrets. By contrast, in myocardium of 6- and 8-week-old ferrets T3 failed to increase protein levels of α1 and α3. To determine whether elevated plasma levels of TH during development plays a role in the transition, mature ferrets were first made hypothyroid before TH treatment. In these hypothyroid ferrets expression of the α3 isoform remained unresponsive to TH (T4, 0.5 mg/kg for 7 days, s.c.). The transition from TH-responsive to TH-unresponsive appears to be isoform-specific because in skeletal muscle of 8-week-old ferrets and in hypothyroid ferrets the α2 isoform is upregulated by TH. Finally, there appears to be functional thyroid hormone receptors throughout development because in each age group TH effectively induced expression of α-MHC in the myocardium. In conclusion, these findings demonstrate that expression of α3 isoform in the myocardium of newborn ferret is responsive to TH; however, the responsiveness terminates between 3- and 6-weeks of age. Neither elevated endogenous TH level nor a lack of functional thyroid hormone receptor appears to be responsible for the transition from TH-responsive to TH-unresponsive.     

Evaluation of 3,4-dihydroquinazoline-2(1H)-thiones as inhibitors of α-MSH-induced melanin production in melanoma B16 cells

Novel 3,4-dihydroquinazoline-2(1H)-thiones (QNTs) 1 were found to be potent inhibitors of α-MSH-induced melanin production. The effect of QNTs to inhibit melanin formation in B16 melanoma cells was screened in the presence of α-MSH. In defining the mechanism of activity, the effects on tyrosinase activity, on tyrosinase synthesis and on the depigmentation of melanin were evaluated. QNTs did not affect the catalytic activity of tyrosinase, but rather acted as an inhibitor of tyrosinase synthesis.     

Angiotensin II promotes differentiation of mouse embryonic stem cells to smooth muscle cells through PI3-kinase signaling pathway and NF-κB

Embryonic stem cells (ES cells), the pluripotent derivatives of the inner cell mass from blastocysts, have the capacity for unlimited growth, self-renewal and differentiation toward all types of somatic cells. Angiotensin II (Ang II), the most important effector peptide of the renin–angiotensin system, is also an angiogenesis factor. However, the potential impact of Ang II on ES cell differentiation is still unknown. In the present study, we have successfully induced the differentiation of ES cells into smooth muscle cells (SMCs) on collagen IV. Interestingly, incubation of ES cells with Ang II further promoted SMC differentiation from ES cells, which was abolished by prior treatment with Ang II type 1 (AT1) receptor antagonist losartan, but not Ang II type 2 (AT2) receptor antagonist PD123319. Moreover, we found that, in parallel with SMC specific-marker induction, the expression levels of phosphoAkt and NF-Kappa B (NF-κB) p50 were up-regulated by Ang II. Importantly, addition of phosphoinositide-3 kinase (PI3K) inhibitor LY294002 led to a marked inhibition of Ang II induced SMC specific markers, phosphoAkt and NF-κB p50 expression. Furthermore, NF-κB inhibitor BAY11-7082 can inhibit Ang II induced expression of SMC specific markers. Thus, we demonstrate for the first time that Ang II plays a promotive role in the stage of ES cell differentiation to SMCs through AT1 receptor. We further confirmed that PI3K/Akt signaling pathway and NF-κB play key roles in this process.     

Gene transfer of a hybrid interleukin-1β gene to B16 mouse melanoma recruits leucocyte subsets and reduces tumour growth in vivo

 Interleukin(IL)-1 differs from most other cytokines in its lack of a signal sequence. This results in intracellular retention of the immature proform. The release of IL-1 has been shown to be restricted predominantly to activated monocytes and macrophages and to be associated with apoptosis of the producer cell. These features have limited the investigation of IL-1 in early immune responses. In order to study the biological effects of local IL-1β release during an antitumour immune response, we used B16 mouse melanoma cells transduced with mature human IL-1β cDNA constructs. To obtain a released form of human IL-1β (ssIL-1β), the signal sequence from the related IL-1 receptor antagonist was ligated to the cDNA that encoded the mature form of IL-1β. When cells of the poorly immunogenic B16 melanoma cell line were transduced with IL-1β by retroviral infection, high levels of the protein were detected intracellularly, whereas cells transduced with IL-1β containing the signal sequence secreted most of their protein. The in vitro growth of the melanoma cells was unaffected by the IL-1β or ssIL-1β gene transfer. In contrast, the in vivo subcutaneous tumour growth of the ssIL-1β-transduced B16 cells in syngeneic C57BL/6 mice was significantly reduced compared with the IL-1β- and the mock-transduced controls. Immunohistochemical analysis revealed the infiltration of macrophages to be strong in B16/ssIL-1β, moderate in B16/IL-1β and minimal in control tumours. Furthermore, a moderate infiltration of CD4⁺ cells and of scattered dendritic cells was detected in B16/ssIL-1β tumours whereas very few or no CD4⁺ cells and dendritic cells were seen in the B16/IL-1β or control tumours. Following in vivo growth, all the tumours up-regulated ICAM-1 on their cell surfaces. However, the percentage of ICAM-1-expressing cells was two- to fourfold higher in B16/ssIL-1β tumours compared to the control. The data suggest that IL-1β acts in vivo, either directly or indirectly, as a chemotactic factor for monocytes, T helper cells and dendritic cells. This supports IL-1β having a regulatory effect on tumour growth when locally released in the tumour area. Received: 12 November 1996 / Accepted: 6 May 1997     

Autophagy facilitates major histocompatibility complex class I expression induced by IFN-γ in B16 melanoma cells

The reduction or loss of MHC-I antigen surface expression in human and murine tumor cells is partly attributable to the dysregulation of various components of the MHC-I antigen-processing machinery. Accumulating evidence suggests that autophagy, besides its vital role in maintaining the cellular homeostasis, plays an important role in MHC-II surface expression. Here, we report that autophagy is a negative regulator of MHC-I antigen expression in B16 melanoma cells; however, in the presence of IFN-γ, it is converted to a positive regulator. We show that autophagy not only participates in the degradation of MHC-I antigen but also plays a role in the generation of MHC-I-binding peptides. For these two processes, IFN-γ interferes with MHC-I antigen degradation, rather than affecting peptide generation. Using B16 melanoma mouse model, we further show that autophagy may enhance the cytolysis of CTL to melanoma cells at the early stage of melanoma, but impairs the cytolysis at the late stage. Such different consequences may be explained by the different levels of IFN-γ during tumor progression. Taken together, our findings demonstrate that autophagy is involved in the regulation of MHC-I antigen expression, through which autophagy can play different roles in tumor immunity.     

Methylation of elongation factor 1α in mouse 3T3B and 3T3BSV40 cells

Two-dimensional gel electrophoretic (NEPHGE) analysis of proteins from mouse 3T3B and 3T3B/SV40 cells labelled with [methyl-³H]methionine in the presence of cycloheximide have revealed that the elongation factor 1α (EF-1α) in these cells is methylated and that the extent of methylation is higher in the SV40 transformed cell type. It is suggested that methylation may account for differences in growth properties for the different cell types.     

Latent HIV-1 infection of resting CD4⁺ T cells in the humanized Rag2⁻/⁻ γc⁻/⁻ mouse

Persistent human immunodeficiency virus type 1 (HIV-1) infection of resting CD4⁺ T cells, unaffected by antiretroviral therapy (ART), provides a long-lived reservoir of HIV infection. Therapies that target this viral reservoir are needed to eradicate HIV-1 infection. A small-animal model that recapitulates HIV-1 latency in resting CD4⁺ T cells may accelerate drug discovery and allow the rational design of nonhuman primate (NHP) or human studies. We report that in humanized Rag2^−/− γ_c^−/− (hu-Rag2^−/− γ_c^−/−) mice, as in humans, resting CD4⁺ T cell infection (RCI) can be quantitated in pooled samples of circulating cells and tissue reservoirs (e.g., lymph node, spleen, bone marrow) following HIV-1 infection with the CCR5-tropic JR-CSF strain and suppression of viremia by ART. Replication-competent virus was recovered from pooled resting CD4⁺ T cells in 7 of 16 mice, with a median frequency of 8 (range, 2 to 12) infected cells per million T cells, demonstrating that HIV-1 infection can persist despite ART in the resting CD4⁺ T cell reservoir of hu-Rag2^−/− γ_c^−/− mice. This model will allow rapid preliminary assessments of novel eradication approaches and combinatorial strategies that may be challenging to perform in the NHP model or in humans, as well as a rigorous analysis of the effect of these interventions in specific anatomical compartments.     

The immunoregulatory role of CD4⁺ FoxP3⁺ CD25⁻ regulatory T cells in lungs of mice infected with Bordetella pertussis

The identification of regulatory T (Treg) cells was originally based on CD25 expression; however, CD25 is also expressed by activated effector T cells. FoxP3 is a more definitive marker of Treg cells, and CD4(+) FoxP3(+) CD25(+) T cells are considered the dominant natural Treg (nTreg) population. It has been suggested that certain CD4(+) FoxP3(+) Treg cells do not express CD25. In this study, we used a murine model of respiratory infection with Bordetella pertussis to examine the role of Treg cells in protective immunity in the lung. We first demonstrated that CD4(+) FoxP3(+) CD25(-) cells are the dominant Treg population in the lung, gut and liver. Pre-activated lung CD4(+) FoxP3(+) CD25(-) cells suppressed CD4(+) effector T cells in vitro, which was partly mediated by IL-10 and not dependent on cell contact. Furthermore, CD4(+) FoxP3(+) CD25(-) IL-10(+) T cells were found in the lungs of mice at the peak of infection with B. pertussis. The rate of bacterial clearance was not affected by depletion of CD25(+) cells or in IL-10-deficient (IL-10(-/-) ) mice, but was compromised in CD25-depleted IL-10(-/-) mice. Our findings suggest that IL-10-producing CD4(+) FoxP3(+) CD25(-) T cells represent an important regulatory cell in the lung.