FAM175B promotes apoptosis by inhibiting ATF4 ubiquitination in esophageal squamous cell carcinoma.

FAM175B is a reported regulator of p53 and suppresses tumorigenesis in numerous types of cancer, but very little is known about its function in esophageal squamous cell carcinomas (ESCCs), almost 70% of which exhibit mutations in p53. Here, we report that FAM175B expression is downregulated in high-grade intraepithelial neoplasia (t = 2.44, P = 0.031) and ESCC (t = 5.664, P < 0.001) tissues relative to that in adjacent normal esophageal tissues. Exogenous expression of FAM175B in ESCC cells resulted in a decrease in proliferation rate, inhibition of colony formation, and an increase in apoptosis rate. Knockdown of FAM175B produced the opposite results. Furthermore, confocal microscopy and coimmunoprecipitation assay showed that Activating transcription factor 4 (ATF4) colocalized and interacted with FAM175B. Ubiquitination assays revealed that FAM175B inhibited ubiquitin-dependent ATF4 degradation and elevated ATF4 protein level. Finally, luciferase reporter experiments further clarified that FAM175B promoted CHOP expression in an ATF4-dependent manner. Accordingly, the proapoptotic activity of FAM175B was significantly rescued by treatment with si-ATF4 and the CHOP inhibitor 4-PBA. In summary, FAM175B inhibited ATF4 ubiquitination and promoted ESCC cell apoptosis in a p53-independent manner. FAM175B expression loss may be an early diagnostic biomarker in ESCC patients.

Correlation Between NKG2DL Expression and Antitumor Effect of Protein-bound Polysaccharide-K in Tumor-bearing Mouse Models.

BACKGROUND/AIM: We investigated the relationship between the expression of natural killer group 2, member D ligands (NKG2DLs) and the antitumor effects of protein-bound polysaccharide-K (PSK). MATERIALS AND METHODS: PSK was administered to evaluate its effectiveness against tumor growth. The expression of Rae-1 and H60 were analyzed in multiple cell lines. RESULTS: PSK showed the highest antitumor effects in mice implanted with cells expressing neither Rae-1 nor H60. PSK had little antitumor effect in mice implanted with cells expressing both Rae-1 and H60. A correlation between the expression of NKG2DLs and the antitumor effect of PSK was observed. After PSK administration, INF-γ production in CD8+ T cells increased in mice with cells expressing neither Rae-1 nor H60, but did not change in mice implanted with cells expressing both Rae-1 and H60. CONCLUSION: We demonstrated that the expression of NKG2DLs affects tumor immunity and the efficacy of immuno therapy in tumor-bearing mouse model.

The Hippo Pathway Targets Rae1 to Regulate Mitosis and Organ Size and to Feed Back to Regulate Upstream Components Merlin, Hippo, and Warts.

Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. Here we identify Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts cyclin B levels and organ size while Rae1 over-expression increases cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of cyclin B and organ size; reducing Rae1 blocks cyclin B accumulation and suppresses overgrowth caused by Hippo Pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue "synthetic lethality" phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, we propose that Rae1 also acts in a feedback circuit to regulate pathway homeostasis.

Heterogeneity of Matrin 3 in the developing and aging murine central nervous system.

Mutations in the MATR3 gene encoding the nucleotide binding protein Matrin 3 have recently been identified as causing a subset of familial amyotrophic lateral sclerosis (fALS) and more rarely causing distal myopathy. Translating the identification of MATR3 mutations into an understanding of disease pathogenesis and the creation of mouse models requires a complete understanding of normal Matrin 3 levels and distribution in vivo. Consequently, we examined the levels of murine Matrin 3 in body tissues and regions of the central nervous system (CNS). We observed a significant degree of variability in Matrin 3 protein levels among different tissues of adult animals, with the highest levels found in reproductive organs and the lowest in muscle. Within the adult CNS, Matrin 3 levels were lowest in spinal cord. Further, we found that Matrin 3 declines significantly in CNS through early development and young adulthood before stabilizing. As previously reported, antibodies to Matrin 3 primarily stain nuclei, but the intensity of staining was not uniform in all nuclei. The low levels of Matrin 3 in spinal cord and muscle could mean that that these tissues are particularly vulnerable to alterations in Matrin 3 function. Our study is the first to characterize endogenous Matrin 3 in rodents across the lifespan, providing the groundwork for deciphering disease mechanisms and developing mouse models of MATR3-linked ALS. J. Comp. Neurol. 524:2740-2752, 2016. © 2016 Wiley Periodicals, Inc.

Proinflammatory Proteins S100A8/S100A9 Activate NK Cells via Interaction with RAGE.

S100A8/A9, a proinflammatory protein, is upregulated in inflammatory diseases, and also has a tumor-promoting activity by the recruitment of myeloid cells and tumor cell invasion. However, whether the expression of S100A8/A9 in tumors predicts a good or poor prognosis is controversial in the clinical setting. In this study, to clarify the in vivo role of S100A8/A9 in the tumor microenvironment, we s.c. inoculated Pan02 cells stably expressing S100A8 and S100A9 proteins (Pan02-S100A8/A9) in syngeneic C57BL/6 mice. Unexpectedly, after small tumor nodules were once established, they rapidly disappeared. Flow cytometry showed that the number of NK cells in the tumors was increased, and an administration of anti-asialoGM1 Ab for NK cell depletion promoted the growth of Pan02-S100A8/A9 s.c. tumors. Although the S100A8/A9 proteins alone did not change the IFN-γ expression of NK cells in vitro, a coculture with Pan02 cells, which express Rae-1, induced IFN-γ production, and Pan02-S100A8/A9 cells further increased the number of IFN-γ(+) NK cells, suggesting that S100A8/A9 enhanced the NK group 2D ligand-mediated intracellular activation pathway in NK cells. We then examined whether NK cell activation by S100A8/A9 was via their binding to receptor of advanced glycation end product (RAGE) by using the inhibitors. RAGE antagonistic peptide and anti-RAGE Ab inhibited the IFN-γ production of NK cells induced by S100A8/A9 proteins, and an administration of FPS-ZM1, a RAGE inhibitor, significantly enhanced the in vivo growth of Pan02-S100A8/A9 tumors. We thus found a novel activation mechanism of NK cells via S100A8/A9-RAGE signaling, which may open a novel perspective on the in vivo interaction between inflammation and innate immunity.

Expression of nuclear matrix proteins binding matrix attachment regions in prostate cancer. PARP-1: New player in tumor progression.

Prostate cancer (PCa) displays infrequent point mutations, whereas genomic rearrangements are highly prevalent. In eukaryotes, the genome is compartmentalized into chromatin loop domains by the attachment to the nuclear matrix (NM), and it has been demonstrated that several recombination hot spots are situated at the base of loops. Here, we have characterized the binding between NM proteins and matrix attachment regions (MARs) in PCa. Nontumor and 44 PCa tissues were analyzed. More aggressive tumors were characterized by an increase in the complexity of the NM protein patterns that was synchronous with a decrease in the number of proteins binding the MAR sequences. PARP-1 was the protein that showed the most evident changes. The expression of the PARP-1 associated with NM increased and it was dependent on tumor aggressiveness. Immunohistochemical analysis showed that the protein was significantly overexpressed in tumor cells. To explore the role of PARP-1 in PCa progression, PCa cells were treated with the PARP inhibitor, ABT-888. In androgen-independent PC3 cells, PARP inhibition significantly decreased cell viability, migration, invasion, chromatin loop dimensions and histone acetylation. Collectively, our study provides evidence that MAR-binding proteins are involved in the development and progression of PCa. PARP could play a key role in the compartmentalization of chromatin and in the development of the more aggressive phenotype. Thus, PARP can no longer be viewed only as an enzyme involved in DNA repair, but that its role in chromatin modulation could provide the basis for a new therapeutic approach to the treatment of PCa.

Combination doxorubicin and interferon-α therapy stimulates immunogenicity of murine pancreatic cancer Panc02 cells via up-regulation of NKG2D ligands and MHC class I.

BACKGROUND: Pancreatic adenocarcinoma is a malignant gastrointestinal cancer with significant morbidity and mortality. Despite severe side effects of chemotherapy, the use of immunotherapy combined with chemotherapy has emerged as a common clinical treatment. In this study, we investigated the efficacy of the combined doxorubicin and interferon-α (IFN-α) therapy on murine pancreatic cancer Panc02 cells in vitro and in vivo and underlying mechanisms. MATERIALS AND METHODS: A Panc02-bearing mouse model was established to determine whether doxorubicin and interferon-α (IFN-α) could effectively inhibit tumor growth in vivo. Cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) was evaluated using a standard LDH release assay. To evaluate the relevance of NK cells and CD8 T cells to the combination therapy-mediated anti-tumor effects, they were depleted in tumor-bearing mice by injecting anti-asialo-GM-1 antibodies or anti-CD8 antibodies, respectively. Finally, the influence of doxorubicin+interferon-α (IFN-α) on the ligands of NK and T cells was assessed by flow cytometry. RESULTS: The combination therapy group demonstrated a significant inhibition of growth of Panc02 in vivo, resulting from activated cytotoxicity of NK cells and CTLs. Depleting CD8 T cells or NK cells reduced the anticancer effects mediated by immunochemotherapy. Furthermore, the doxorubicin+IFN-a treatment increased the expression of major histocompatibility complex class I (MHC I) and NKG2D ligands on Panc02 cells, suggesting that the combined therapy may be a potential strategy for enhancing immunogenicity of tumors. All these data indicate that the combination therapy using doxorubicin and interferon-α (IFN-α) may be a potential strategy for treating pancreatic adenocarcinoma.

RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma.

The immunoreceptor NKG2D originally identified in natural killer (NK) cells recognizes ligands that are upregulated on tumor cells. Expression of NKG2D ligands (NKG2DL) is induced by the DNA damage response (DDR), which is often activated constitutively in cancer cells, revealing them to NK cells as a mechanism of immunosurveillance. Here, we report that the induction of retinoic acid early transcript 1 (RAE1) ligands for NKG2D by the DDR relies on a STING-dependent DNA sensor pathway involving the effector molecules TBK1 and IRF3. Cytosolic DNA was detected in lymphoma cell lines that express RAE1 and its occurrence required activation of the DDR. Transfection of DNA into ligand-negative cells was sufficient to induce RAE1 expression. Irf3(+/-);Eµ-Myc mice expressed lower levels of RAE1 on tumor cells and showed a reduced survival rate compared with Irf3(+/+);Eµ-Myc mice. Taken together, our results suggest that genomic damage in tumor cells leads to activation of STING-dependent DNA sensor pathways, thereby activating RAE1 and enabling tumor immunosurveillance.

Activation of cellular immunity and marked inhibition of liver cancer in a mouse model following gene therapy and tumor expression of GM-SCF, IL-21, and Rae-1.

BACKGROUND: Cancer is both a systemic and a genetic disease. The pathogenesis of cancer might be related to dampened immunity. Host immunity recognizes nascent malignant cells - a process referred to as immune surveillance. Augmenting immune surveillance and suppressing immune escape are crucial in tumor immunotherapy. METHODS: A recombinant plasmid capable of co-expressing granulocyte-macrophage colony- stimulating factor (GM-SCF), interleukin-21 (IL-21), and retinoic acid early transcription factor-1 (Rae-1) was constructed, and its effects determined in a mouse model of subcutaneous liver cancer. Serum specimens were assayed for IL-2 and INF-γ by ELISA. Liver cancer specimens were isolated for Rae-1 expression by RT-PCR and Western blot, and splenocytes were analyzed by flow cytometry. RESULTS: The recombinant plasmid inhibited the growth of liver cancer and prolonged survival of tumor-loaded mice. Activation of host immunity might have contributed to this effect by promoting increased numbers and cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) following expression of GM-SCF, IL-21, and Rae-1. By contrast, the frequency of regulatory T cells was decreased, Consequently, activated CTL and NK cells enhanced their secretion of INF-γ, which promoted cytotoxicity of NK cells and CTL. Moreover, active CTL showed dramatic secretion of IL-2, which stimulates CTL. The recombinant expression plasmid also augmented Rae-1 expression by liver cancer cells. Rae-1 receptor expressing CTL and NK cells removed liver cancer. CONCLUSIONS: The recombinant expression plasmid inhibited liver cancer by a mechanism that involved activation of cell-mediated immunity and Rae-1 in liver cancer.

NKG2D blockade facilitates diabetes prevention by antigen-specific Tregs in a virus-induced model of diabetes.

It is thought that viral infections might jeopardize regulatory T cell therapy in type 1 diabetes. Viral infections can lead to surface expression of ligands for the activating NKG2D receptor, such as retinoic acid early transcript 1 (Rae-1), whose expression on beta-cells recruits NKG2D(+) autoreactive CD8(+) T cells. Both in men and mice, autoreactive cytotoxic T cells express NKG2D. We showed that NKG2D expression increased on CD4(+) and CD8(+) T cells during virus-induced diabetes development in the rat insulin promotor (RIP) Lymphocytic Choriomeningitis Virus (LCMV) model. Combination treatment with anti-NKG2D and antigen-specific regulatory T cells (Treg), at doses inefficacious in mono-treatment, synergized to prevent diabetes in 75% of the virus-infected RIP-LCMV mice. Nevertheless, NKG2D blockade alone failed to reverse recent-onset diabetes in non-obese diabetic (NOD) mice, despite downregulation of NKG2D on NK cells in the blood and CD8(+) T cells in the spleen and pancreatic lymph nodes. Our data suggest that blocking the interaction of NKG2D with it ligands is insufficient to protect against diabetes when a strong inflammatory process actively drives NKG2D upregulation, but should be considered to help maintaining Treg functionality during ongoing pancreatic inflammation.

Suppressing T cell motility induced by anti-CTLA-4 monotherapy improves antitumor effects.

A promising strategy for cancer immunotherapy is to disrupt key pathways regulating immune tolerance, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4). However, the determinants of response to anti-CTLA-4 mAb treatment remain incompletely understood. In murine models, anti-CTLA-4 mAbs alone fail to induce effective immune responses to poorly immunogenic tumors but are successful when combined with additional interventions, including local ionizing radiation (IR) therapy. We employed an established model based on control of a mouse carcinoma cell line to study endogenous tumor-infiltrating CD8+ T lymphocytes (TILs) following treatment with the anti-CTLA-4 mAb 9H10. Alone, 9H10 monotherapy reversed the arrest of TILs with carcinoma cells in vivo. In contrast, the combination of 9H10 and IR restored MHC class I-dependent arrest. After implantation, the carcinoma cells had reduced expression of retinoic acid early inducible-1 (RAE-1), a ligand for natural killer cell group 2D (NKG2D) receptor. We found that RAE-1 expression was induced by IR in vivo and that anti-NKG2D mAb blocked the TIL arrest induced by IR/9H10 combination therapy. These results demonstrate that anti-CTLA-4 mAb therapy induces motility of TIL and that NKG2D ligation offsets this effect to enhance TILs arrest and antitumor activity.

CIZ/NMP4 is expressed in B16 melanoma and forms a positive feedback loop with RANKL to promote migration of the melanoma cells.

Tumor metastasis to bone is a serious pathological situation that causes severe pain, and deterioration in locomoter function. However, the mechanisms underlying tumor metastasis is still incompletely understood. CIZ/NMP4 is a nucleocytoplasmic shuttling protein and its roles in tumor cells have not been known. We, therefore, hypothesized the role of CIZ/NMP4 in B16 melanoma cells that metastasize to bone. CIZ/NMP4 is expressed in B16 cells. The CIZ/NMP4 expression levels are correlated to the metastatic activity in divergent types of melanoma cells. Overexpression of CIZ/NMP4 increased B16 cell migration in Trans-well assay. Conversely, siRNA-based knockdown of CIZ/NMP4 suppressed migratory activity of these cells. As RANKL promotes metastasis of tumor cells in bone, we tested its effect on CIZ in melanoma cells. RANKL treatment enhanced CIZ/NMP4 expression. This increase of CIZ by RANKL promoted migration. Conversely, we identified CIZ/NMP4 binding site in the promoter of RANKL. Furthermore, luciferase assay indicated that CIZ/NMP4 overexpression enhanced RANKL promoter activities, revealing a positive feedback loop of CIZ/NMP4 and RANKL in melanoma. These observations indicate that CIZ/NMP4 is critical regulator of metastasis of melanoma cells.

Expression of the RAE-1 family of stimulatory NK-cell ligands requires activation of the PI3K pathway during viral infection and transformation.

Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands.

Toll-like receptor 4 engagement contributes to expression of NKG2D ligands by renal tubular epithelial cells.

BACKGROUND: Engagement of Toll-like receptor (TLR) 4 on intrinsic kidney cells is critical for the full development of renal ischaemia-reperfusion injury (IRI). Effects of TLR signalling in renal parenchymal cells include the production of cytokines, chemokines and other soluble mediators which contribute to local inflammation and leucocyte accumulation. Whether engagement of TLR4 on kidney cells results in additional pro-inflammatory modifications of the renal microenvironment remains to be determined. METHODS: Renal IRI was induced by clamping of the renal pedicles, and expression of NKG2D ligands in mice deficient in TLR4 or its adaptor molecule MyD88, or else pretreated with blocking antibodies against the endogenous TLR4 ligand HMGB1, was compared to that in wild-type mice. Cultures of isolated renal tubular epithelial cells (TECs) from WT, TLR4(-/-) and MyD88(-/-) mice were stimulated with the TLR4 ligand lipopolysaccharide (LPS), or mineral oil occlusion was used to simulate IRI in vitro, prior to determination of NKG2D ligand expression. Chimeric mice lacking TLR4 in either the bone marrow derived or the parenchymal compartment were also subjected to IRI. RESULTS: In this study, we demonstrate a substantial increase in the expression of the NKG2D ligands retinoic acid early inducible-1 (RAE-1), murine ULBP-like transcript 1 (MULT-1) and histocompatibility-60 (H-60) in mouse kidneys during renal IRI. Expression of NKG2D ligands was attenuated in mice deficient in either TLR4 or the adaptor molecule MyD88. Antibody blockade of HMGB1 reduced NKG2D ligand expression by a comparable extent to TLR4 deficiency and did not result in further reduction of NKG2D ligand expression in TLR4(-/-) mice. Isolated TECs from normal mice but not those with defects in the TLR4-MyD88 signalling pathway expressed RAE-1 and MULT-1 upon exposure to LPS and after being subjected to in vitro conditions resembling ischaemia-reperfusion. TLR4 competence in the parenchymal but not the bone marrow-derived compartment was required for RAE-1 up-regulation in mouse kidneys after ischaemia, while TLR4 signalling in both compartments contributed to the intrarenal expression of MULT-1 during IRI. CONCLUSION: Expression of the NKG2D ligands RAE-1 and MULT-1 on kidney cells in response to TLR4 engagement by HMGB1 represents another mechanism by which TLR4 signalling may participate in the pathogenesis of renal IRI.

Separation and identification of differentially expressed nuclear matrix proteins in breast carcinoma forming.

BACKGROUND: Breast carcinoma is one of most prevalent malignant tumors occurring in women. Short of prevention, detection of breast carcinoma at an early, still curable stage would offer the best route to decrease its mortality rates. This highlights the urgent need for suitable biomarkers for early diagnosis and a better understanding of the disease pathogenesis. MATERIAL AND METHODS: NMPs were extracted from normal human breast tissue (Group I), from hyperplastic mammary tissue specimens (Group II), from atypical epithelial hyperplasia specimens (Group III), and from breast carcinoma (Group IV) tissue. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The different NMPs were analyzed in the National Center for Biotechnology Information (NCBI) database with Mascot software. RESULTS: Well-resolved, reproducible 2-DE profiles of human breast tissues were obtained. Average protein spots were 904 +/- 58, 912 +/- 51, 931 +/- 63, 944 +/- 70 in Group I, Group II, Group III, and Group IV, respectively. Several different proteins were analyzed using mass spectrometry and bioinformation. Of these, 12 were well characterized. Compared to Group I, three proteins were up-regulated in Groups II, III, and IV, including Hsp27, prohibitin, and laminA/C. Upregulation was confirmed using Western blotting and immunohistochemical analysis. The correlation of prohibitin expression with clinicopathological features was also investigated. DISCUSSION: The proteins identified in this study may potentially prove to be useful markers for breast carcinoma diagnosis.

Low SAFB levels are associated with worse outcome in breast cancer patients.

The scaffold attachment factors SAFB1 and SAFB2 have been shown to function as estrogen receptor (ERalpha) co-repressors in breast cancer cells, and to affect many cellular processes such as stress response, RNA processing, and apoptosis. SAFB1 and SAFB2 have also been implicated in breast tumorigenesis: Their shared chromosomal locus at 19p13 is frequently lost in breast cancer, mutations have been identified, and overexpression results in growth inhibition. The purpose of this study was to determine SAFB1/SAFB2 protein expression in human breast tumors, to correlate their expression with either natural progression ("prognostic factor") or with response to Tamoxifen ("predictive factor"), and to analyze potential correlations with tumor characteristics. SAFB1/SAFB2 protein were measured by immunoblotting using a pan-SAFB antibody in tumor extracts from patients with long-term clinical follow-up (n = 289), a subset of whom had received no adjuvant systemic therapy after breast cancer surgery (n = 117) and another subset of whom were treated with adjuvant Tamoxifen (n = 172). SAFB levels were correlated with clinico-pathological variables and patient outcome. SAFB levels varied widely, with 25 tumors not expressing detectable levels of SAFB. SAFB expression was significantly correlated with ERalpha, HER-2, bcl-2 and with expression of other ERalpha coregulators such as SRC-3. There was no association between SAFB expression and disease free survival, however, low SAFB expression was significantly associated with worse overall survival in patients who did not receive adjuvant therapy. This study shows that low SAFB protein levels predict poor prognosis of breast cancer patients, suggesting critical functions of SAFB1 and SAFB2 in breast cancer cells.

Chimeric NKG2D receptor-bearing T cells as immunotherapy for ovarian cancer.

Despite advancements in the treatment of ovarian cancer, this disease continues to be a leading cause of cancer death in women. Adoptive transfer of tumor-reactive T cells is a promising antitumor therapy for many cancers. We designed a chimeric receptor linking NKG2D, a natural killer (NK) cell-activating receptor, to the CD3zeta chain of the T-cell receptor to target ovarian tumor cells. Engagement of chimeric NKG2D receptors (chNKG2D) with ligands for NKG2D, which are commonly expressed on tumor cells, leads to T-cell secretion of proinflammatory cytokines and tumor cytotoxicity. In this study, we show that >80% of primary human ovarian cancer samples expressed ligands for NKG2D on the cell surface. The tumor samples expressed MHC class I-related protein A, MICB, and UL-16 binding proteins 1 and 3. ChNKG2D-expressing T cells lysed ovarian cancer cell lines. We show that T cells from ovarian cancer patients that express chNKG2D secreted proinflammatory cytokines when cultured with autologous tumor cells. In addition, we show that chNKG2D T cells can be used therapeutically in a murine model of ovarian cancer. These data indicate that treatment with chNKG2D-expressing T cells is a potential immunotherapy for ovarian cancer.

[Effects of NKG2D and its ligands RAE-1 and H60 on graft-versus-tumor response].

The study was purposed to explore the effects of NKG2D receptor and its ligands RAE-1 and H60 on graft-versus-tumor (GVT) response induced by MHC haploidentical bone marrow/spleen cell transplantation. Female (BALB/c x C57BL/6) F1 mice (CB6F1, H-2K(b/d)) inoculated with H22 cells to develop a solid tumor model were the recipients, and bone marrow mixed with spleen cells of the healthy male C57BL/6 (H-2K(b)) mice were the donor cells. GVT response was observed after transplantation that from donor cells T and NK cells were purged with anti-CD3 and anti-NK monoclonal antibody, and the NKG2D receptor was blocked with anti-NKG2D monoclonal antibody, the expression levels of RAE-1 and H60 mRNA in tumor tissue were measured by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) at different time points after transplantation. The results showed that the GVT response of transplantation was reduced after in vitro depletion of T and NK cells or blocking NKG2D receptor in donor cells of the graft, the expression levels of RAE-1 and H60 mRNA in tumor tissue increased after transplantation of haploidential bone marrow mixed with spleen cells. It is concluded that NKG2D and its ligands RAE-1 and H60 may play important roles in GVT response.

Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer.

BACKGROUND: Improving human nuclear transfer (NT) efficiencies is paramount for the development of patient-specific stem cell lines, although the opportunities remain limited owing to difficulties in obtaining fresh mature oocytes. METHODS: Therefore, the developmental competence of aged, failed-to-fertilize human oocytes as an alternate cytoplasmic source for NT was assessed and compared with use of fresh, ovulation-induced oocytes. To further characterize the developmental potential of aged oocytes, parthenogenetic activation, immunocytochemical analysis of essential microtubule proteins involved in meiotic and mitotic division, and RT-PCR in single oocytes (n = 6) was performed to determine expression of oocyte-specific genes [oocyte-specific histone 1 (H1FOO), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zygote arrest 1 (ZAR1)] and microtubule markers [nuclear mitotic arrest (NuMA), minus-end directed motor protein HSET and the microtubule kinesin motor protein EG5]. RESULTS: For NT, enucleation and fusion rates of aged oocytes were significantly lower compared with fresh oocytes (P < 0.05). Cleavage rates and subsequent development were poor. In addition, parthenote cleavage was low. Immunocytochemical analysis revealed that many oocytes displayed aberrant expression of NuMA and EG5, had disrupted meiotic spindles and tetrapolar spindles. One of the six oocytes misexpressed GDF9, BMP15 and ZAR1. Two oocytes expressed EG5 messenger RNA (mRNA), and HSET and NuMA were not detectable. RT-PCR of mRNA for oocyte specific genes and microtubule markers in single aged oocytes. CONCLUSIONS: Thus, aneuploidy and spindle defects may contribute to poor parthenogenetic development and developmental outcomes following NT.

Nuclear matrix protein expression in prostate cancer: possible prognostic and diagnostic applications.

Different lines of evidence suggest that the nuclear matrix (NM), the protein scaffold of the nucleus, represents a functional unit playing a pivotal role in the spatial and temporal coordination of the events of gene activation. Any change in the gene expression pattern, which occurs during carcinogenesis, may partially depend on an impairment of the regulatory functions of the NM. Therefore, increasing interest has been addressed to the study of NM modifications associated with malignant transformations and to potential clinical applications. Here, recent results on the NM changes in prostate cancer are discussed. Tumor cells are characterized by a more complex NM protein pattern compared to normal tissue: the development of poorly-differentiated tumors is characterized by the expression of proteins that are not present in hyperplastic tissues or in more differentiated tumors. In addition, a few newly-expressed proteins are significantly correlated with the risk of biochemical progression. The potential application of these proteins at the diagnostic and prognostic levels calls for further studies.