Novel Dormancy Mechanism of Castration Resistance in Bone Metastatic Prostate Cancer Organoids.

Advanced prostate cancer (PCa) patients with bone metastases are treated with androgen pathway directed therapy (APDT). However, this treatment invariably fails and the cancer becomes castration resistant. To elucidate resistance mechanisms and to provide a more predictive pre-clinical research platform reflecting tumor heterogeneity, we established organoids from a patient-derived xenograft (PDX) model of bone metastatic prostate cancer, PCSD1. APDT-resistant PDX-derived organoids (PDOs) emerged when cultured without androgen or with the anti-androgen, enzalutamide. Transcriptomics revealed up-regulation of neurogenic and steroidogenic genes and down-regulation of DNA repair, cell cycle, circadian pathways and the severe acute respiratory syndrome (SARS)-CoV-2 host viral entry factors, ACE2 and TMPRSS2. Time course analysis of the cell cycle in live cells revealed that enzalutamide induced a gradual transition into a reversible dormant state as shown here for the first time at the single cell level in the context of multi-cellular, 3D living organoids using the Fucci2BL fluorescent live cell cycle tracker system. We show here a new mechanism of castration resistance in which enzalutamide induced dormancy and novel basal-luminal-like cells in bone metastatic prostate cancer organoids. These PDX organoids can be used to develop therapies targeting dormant APDT-resistant cells and host factors required for SARS-CoV-2 viral entry.

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient's bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Exosomes: Versatile Nano Mediators of Immune Regulation.

One of many types of extracellular vesicles (EVs), exosomes are nanovesicle structures that are released by almost all living cells that can perform a wide range of critical biological functions. Exosomes play important roles in both normal and pathological conditions by regulating cell-cell communication in cancer, angiogenesis, cellular differentiation, osteogenesis, and inflammation. Exosomes are stable in vivo and they can regulate biological processes by transferring lipids, proteins, nucleic acids, and even entire signaling pathways through the circulation to cells at distal sites. Recent advances in the identification, production, and purification of exosomes have created opportunities to exploit these structures as novel drug delivery systems, modulators of cell signaling, mediators of antigen presentation, as well as biological targeting agents and diagnostic tools in cancer therapy. This review will examine the functions of immunocyte-derived exosomes and their roles in the immune response under physiological and pathological conditions. The use of immunocyte exosomes in immunotherapy and vaccine development is discussed.

Complete androgen insensitivity syndrome caused by c.1769-1G > C mutation and activation of a cryptic splice acceptor site in the androgen receptor gene.

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of sex development (46,XY DSD). This syndrome is an X-linked recessive genetic disease characterized by resistance to the actions of androgens in an individual with a male karyotype and it is caused by mutations in the androgen receptor (AR) gene. We evaluated two siblings with primary amenorrhea, normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the AR gene revealed a splice acceptor site mutation in intron 2 (c.1769-1G > C). The analysis of mRNA showed that this mutation resulted in the activation of a cryptic splice acceptor site located in intron 2 and in the synthesis of an aberrant mRNA transcript with 69 nucleotides insertion between exon 2 and exon 3, leading to an insertion of 23 amino acids in the AR protein instead of generating a premature termination codon. The additional 23 amino acids insertion affects AR intracellular trafficking by impairing its translocation from the cytoplasm to the nucleus after hormone stimulation. The c.1769-1G > C mutation provides new insights into the molecular mechanism involved in splicing defects and expands the spectrum of mutations associated with the androgen insensitivity syndrome.

Regulation of Natural Killer Cell Function by STAT3.

Natural killer (NK) cells, key members of a distinct hematopoietic lineage, innate lymphoid cells, are not only critical effectors that mediate cytotoxicity toward tumor and virally infected cells but also regulate inflammation, antigen presentation, and the adaptive immune response. It has been shown that NK cells can regulate the development and activation of many other components of the immune response, such as dendritic cells, which in turn, modulate the function of NK cells in multiple synergistic feed back loops driven by cell-cell contact, and the secretion of cytokines and chemokines that control effector function and migration of cells to sites of immune activation. The signal transducer and activator of transcription (STAT)-3 is involved in driving almost all of the pathways that control NK cytolytic activity as well as the reciprocal regulatory interactions between NK cells and other components of the immune system. In the context of tumor immunology, NK cells are a first line of defense that eliminates pre-cancerous and transformed cells early in the process of carcinogenesis, through a mechanism of "immune surveillance." Even after tumors become established, NK cells are critical components of anticancer immunity: dysfunctional NK cells are often found in the peripheral blood of cancer patients, and the lack of NK cells in the tumor microenvironment often correlates to poor prognosis. The pathways and soluble factors activated in tumor-associated NK cells, cancer cells, and regulatory myeloid cells, which determine the outcome of cancer immunity, are all critically regulated by STAT3. Using the tumor microenvironment as a paradigm, we present here an overview of the research that has revealed fundamental mechanisms through which STAT3 regulates all aspects of NK cell biology, including NK development, activation, target cell killing, and fine tuning of the innate and adaptive immune responses.

Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer.

OBJECTIVE: Bone metastasis occurs in up to 90% of men with advanced prostate cancer and leads to fractures, severe pain and therapy-resistance. Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients. Thus, the special environment of the bone makes the disease more complicated and incurable. A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments. The microstructural changes in the femurs of mice implanted with PCSD1, a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen, were determined. METHODS: Quantitative micro-computed tomography (micro-CT) and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone (Control) into the right femurs of Rag2-/-γc-/- male mice. RESULTS: Bone lesions formed only in femurs of mice injected with PCSD1 cells. Bone volume (BV) was significantly decreased at the proximal and distal ends of the femurs (p < 0.01) whereas BV (p < 0.05) and bone shaft diameter (p < 0.01) were significantly increased along the femur shaft. CONCLUSION: PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur, and, in contrast, induced aberrant bone formation leading to osteoblastic lesions along the femur shaft. Therefore, the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion. Our approach can be used to determine if different bone regions support more therapy resistant tumor growth, thus, requiring novel treatments.

Suppressor of cytokine signaling (SOCS) genes are silenced by DNA hypermethylation and histone deacetylation and regulate response to radiotherapy in cervical cancer cells.

Suppressor of cytokine signaling (SOCS) family is an important negative regulator of cytokine signaling and deregulation of SOCS has been involved in many types of cancer. All cervical cancer cell lines tested showed lower expression of SOCS1, SOCS3, and SOCS5 than normal tissue or cell lines. The immunohistochemistry result for SOCS proteins in human cervical tissue also confirmed that normal tissue expressed higher level of SOCS proteins than neighboring tumor. Similar to the regulation of SOCS in other types of cancer, DNA methylation contributed to SOCS1 downregulation in CaSki, ME-180, and HeLa cells. However, the expression of SOCS3 or SOCS5 was not recovered by the inhibition of DNA methylation. Histone deacetylation may be another regulatory mechanism involved in SOCS1 and SOCS3 expression, however, SOCS5 expression was neither affected by DNA methylation nor histone deacetylation. Ectopic expression of SOCS1 or SOCS3 conferred radioresistance to HeLa cells, which implied SOCS signaling regulates the response to radiation in cervical cancer. In this study, we have shown that SOCS expression repressed by, in part, epigenetically and altered SOCS1 and SOCS3 expression could contribute to the radiosensitive phenotype in cervical cancer.

PCSD1, a new patient-derived model of bone metastatic prostate cancer, is castrate-resistant in the bone-niche.

INTRODUCTION: Prostate cancer bone metastasis occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with therapy resistance and rapid decline. Androgen deprivation therapy (ADT) is standard of care for advanced prostate cancer, however, bone metastatic prostate cancer (PCa) often becomes resistant to ADT. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. Here we report the castrate resistant growth in the bone niche of PCSD1, a patient-derived intra-femoral xenograft model of prostate bone metastatic cancer treated with the anti-androgen, bicalutamide. METHODS: PCSD1 bone-niche model was derived from a human prostate cancer femoral metastasis resected during hemiarthroplasty and serially transplanted into Rag2(-/-); γ c(-/-) mice intra-femorally (IF) or sub-cutaneously (SC). At 5 weeks post-transplantation mice received bicalutamide or vehicle control for 18 days. Tumor growth of PCSD1 was measured with calipers. PSA expression in PCSD1 xenograft tumors was determined using quantitative RT-PCR and immunohistochemistry. Expression of AR and PSMA, were also determined with qPCR. RESULTS: PCSD1 xenograft tumor growth capacity was 24 fold greater in the bone (intra-femoral, IF) than in the soft tissue (sub-cutaneous, SC) microenvironment. Treatment with the anti-androgen, bicalutamide, inhibited tumor growth in the sub-cutaneous transplantation site. However, bicalutamide was ineffective in suppressing PCSD1 tumor growth in the bone-niche. Nevertheless, bicalutamide treatment of intra-femoral tumors significantly reduced PSA expression (p < = 0.008) and increased AR (p < = 0.032) relative to control. CONCLUSIONS: PCSD1 tumors were castrate resistant when growing in the bone-niche compared to soft tissue. Bicalutamide had little effect on reducing tumor burden in the bone yet still decreased tumor PSA expression and increased AR expression, thus, this model closely recapitulated castrate-resistant, human prostate cancer bone metastatic disease. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study bone metastatic disease and for pre-clinical drug development of novel therapies for inhibiting therapy resistant prostate cancer growth in the bone-niche.

Natural killer cells preferentially target cancer stem cells; role of monocytes in protection against NK cell mediated lysis of cancer stem cells.

Mounting effective anti-tumor immune responses by cytotoxic effectors is important for the clearance of tumors. However, accumulated evidence suggests that the cytotoxic function of immune effectors is largely suppressed in the tumor microenvironment by a number of distinct effectors and their secreted factors. The aims of this review are to provide a rationale and potential mechanism for immunosuppression in cancer, and to demonstrate the significance of such immunosuppression in cellular differentiation and tissue regeneration in pathological conditions, and progression of cancer. We have recently shown that increased NK cell function was seen when they were cultured with primary oral squamous carcinoma stem cells (OSCSCs) as compared to their more differentiated oral squamous carcinoma cells (OSCCs). In addition, human embryonic stem cells (hESCs), Mesenchymal Stem Cells (hMSCs), dental pulp stem cells (hDPSCs) and induced pluripotent stem cells (hiPSCs) were significantly more susceptible to NK cell mediated cytotoxicity than their differentiated counterparts or parental cells from which they were derived. We have also reported that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 augmented NK cell function significantly. Total population of monocytes and those depleted of CD16(+) subsets were able to substantially prevent NK cell mediated lysis of OSCSCs, MSCs and DPSCs. Taken together, our results suggest that stem cells are significant targets of the NK cell cytotoxicity. The concept of split anergy in NK cells and its contribution to tissue repair and regeneration and in tumor resistance and progression will be discussed in this review. Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation at the site of the tumor for specific elimination of cancer stem cells.

A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions.

UNLABELLED: Prostate cancer metastasizes to bone in the majority of patients with advanced disease leading to painfully debilitating fractures, spinal compression and rapid decline. In addition, prostate cancer bone metastases often become resistant to standard therapies including androgen deprivation, radiation and chemotherapy. There are currently few models to elucidate mechanisms of interaction between the bone microenvironment and prostate cancer. It is, thus, essential to develop new patient-derived, orthotopic models. Here we report the development and characterization of PCSD1 (Prostate Cancer San Diego 1), a novel patient-derived intra-femoral xenograft model of prostate bone metastatic cancer that recapitulates mixed osteolytic and osteoblastic lesions. METHODS: A femoral bone metastasis of prostate cancer was removed during hemiarthroplasty and transplanted into Rag2(-/-);γc(-/-) mice either intra-femorally or sub-cutaneously. Xenograft tumors that developed were analyzed for prostate cancer biomarker expression using RT-PCR and immunohistochemistry. Osteoblastic, osteolytic and mixed lesion formation was measured using micro-computed tomography (microCT). RESULTS: PCSD1 cells isolated directly from the patient formed tumors in all mice that were transplanted intra-femorally or sub-cutaneously into Rag2(-/-);γc(-/-) mice. Xenograft tumors expressed human prostate specific antigen (PSA) in RT-PCR and immunohistochemical analyses. PCSD1 tumors also expressed AR, NKX3.1, Keratins 8 and 18, and AMACR. Histologic and microCT analyses revealed that intra-femoral PCSD1 xenograft tumors formed mixed osteolytic and osteoblastic lesions. PCSD1 tumors have been serially passaged in mice as xenografts intra-femorally or sub-cutaneously as well as grown in culture. CONCLUSIONS: PCSD1 xenografts tumors were characterized as advanced, luminal epithelial prostate cancer from a bone metastasis using RT-PCR and immunohistochemical biomarker analyses. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely resembled the bone lesions in the patient. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study metastatic disease in the bone and to develop novel therapies for inhibiting prostate cancer growth in the bone-niche.

AID-induced genotoxic stress promotes B cell differentiation in the germinal center via ATM and LKB1 signaling.

During an immune response, B cells undergo rapid proliferation and activation-induced cytidine deaminase (AID)-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here, we report that exogenous and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.

Differential effect of ionizing radiation exposure on multipotent and differentiation-restricted bone marrow mesenchymal stem cells.

Debilitating effects of bone marrow from ionizing radiation exposure has been well established for hematopoietic stem cells; however, radiation toxicity of mesenchymal stem cells (MSCs) has been controversial. The present study addressed if ionizing radiation exposure differently affected bone marrow MSCs with various differentiation commitments. Mouse bone-marrow-derived MSCs, D1 cells of early passages (≤ 5 passages; p5) maintained the complete characteristics of multipotent MSCs, whereas, after ≥ 45 passages (p45) the differentiation capability of D1 cells became partially restricted. Both p5 and p45 D1 cells were subjected to single dose irradiation by radioactive isotope (137)Cs. Radiation treatment impaired cell renewal and differentiation activities of p5 D1 cells; however, p45 D1 cells were less affected. Radiation treatment upregulated both pro- and anti-apoptotic genes of p5 D1 cells in a dose-dependent manner, potentially resulting in the various apoptosis thresholds. It was found that constitutive as well as radiation-induced phosphorylation levels of histone H2AX was significantly higher in p45 D1 cells than in p5 D1 cells. The increased repair activity of DNA double-strand breakage may play a role for p45 D1 cells to exhibit the relative radioresistance. In conclusion, the radiation toxicity predominantly affecting multipotent MSCs may occur at unexpectedly low doses, which may, in part, contribute to the catabolic pathology of bone tissue.

Strategies to rescue mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) from NK cell mediated cytotoxicity.

BACKGROUND: The aim of this paper is to study the function of allogeneic and autologous NK cells against Dental Pulp Stem Cells (DPSCs) and Mesenchymal Stem Cells (MSCs) and to determine the function of NK cells in a three way interaction with monocytes and stem cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that freshly isolated untreated or IL-2 treated NK cells are potent inducers of cell death in DPSCs and MSCs, and that anti-CD16 antibody which induces functional split anergy and apoptosis in NK cells inhibits NK cell mediated lysis of DPSCs and MSCs. Monocytes co-cultured with either DPSCs or MSCs decrease lysis of stem cells by untreated or IL-2 treated NK cells. Monocytes also prevent NK cell apoptosis thereby raising the overall survival and function of NK cells, DPSCs or MSCs. Both total population of monocytes and those depleted of CD16(+) subsets were able to prevent NK cell mediated lysis of MSCs and DPSCs, and to trigger an increased secretion of IFN-gamma by IL-2 treated NK cells. Protection of stem cells from NK cell mediated lysis was also seen when monocytes were sorted out from stem cells before they were added to NK cells. However, this effect was not specific to monocytes since the addition of T and B cells to stem cells also protected stem cells from NK cell mediated lysis. NK cells were found to lyse monocytes, as well as T and B cells. CONCLUSION/SIGNIFICANCE: By increasing the release of IFN-gamma and decreasing the cytotoxic function of NK cells monocytes are able to shield stem cells from killing by the NK cells, resulting in an increased protection and differentiation of stem cells. More importantly studies reported in this paper indicate that anti-CD16 antibody can be used to prevent NK cell induced rejection of stem cells.

Dexamethasone induces dysferlin in myoblasts and enhances their myogenic differentiation.

Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating dysferlinopathy, a rare muscular dystrophy caused by loss of dysferlin. We sought to understand the molecular basis for this disparity by studying the effects of a glucocorticoid on differentiation of the myoblast cell line, C2C12, and dysferlin-deficient C2C12s. We found that pharmacologic doses of dexamethasone enhanced the myogenic fusion efficiency of C2C12s and increased the induction of dysferlin, along with specific myogenic transcription factors, sarcolemmal and structural proteins. In contrast, the dysferlin-deficient C2C12 cell line demonstrated a reduction in long myotubes and early induction of particular muscle differentiation proteins, most notably, myosin heavy chain. Dexamethasone partially reversed the defect in myogenic fusion in the dysferlin-deficient C2C12 cells. We hypothesize that a key therapeutic benefit of glucocorticoids may be the up-regulation of dysferlin as an important component of glucocorticoid-enhanced myogenic differentiation.

N-acetyl cysteine mediates protection from 2-hydroxyethyl methacrylate induced apoptosis via nuclear factor kappa B-dependent and independent pathways: potential involvement of JNK.

The mechanisms by which resin based materials induce adverse effects in patients have not been completely elucidated. Here we show that 2-hydroxyethyl methacrylate (HEMA) induces apoptotic cell death in oral keratinocytes. Functional loss and cell death induced by HEMA was significantly inhibited in the presence of N-acetyl cysteine (NAC) treatment. NAC also prevented HEMA mediated decrease in vascular endothelial growth factor secretion. The protective effect of NAC was partly related to its ability to induce NF-kappaB in the cells, since HEMA mediated inhibition of nuclear NF-kappaB expression and function was significantly blocked in the presence of NAC treatment. Moreover, blocking of nuclear translocation of NF-kappaB in oral keratinocytes sensitized these cells to HEMA mediated apoptosis. In addition, since NAC was capable of rescuing close to 50% of NF-kappaB knockdown cells from HEMA mediated cell death, there is, therefore, an NF-kappaB independent pathway of protection from HEMA mediated cell death by NAC. NAC mediated prevention of HEMA induced cell death in NF-kappaB knockdown cells was correlated with a decreased induction of c-Jun N-terminal kinase (JNK) activity since NAC inhibited HEMA mediated increase in JNK levels. Furthermore, the addition of a pharmacologic JNK inhibitor to HEMA treated cells prevented cell death and restored NF-kappaB knockdown cell function significantly. Therefore, NAC protects oral keratinocytes from the toxic effects of HEMA through NF-kappaB dependent and independent pathways. Moreover, our data suggest the potential involvement of JNK pathway in NAC mediated protection.

Regulation of IGFBP6 gene and protein is mediated by the inverse expression and function of c-jun N-terminal kinase (JNK) and NFkappaB in a model of oral tumor cells.

The aim of this study is to identify potential gene and protein targets when nuclear factor kappa B (NFkappaB) and c-jun N-terminal kinase (JNK) were inversely expressed in oral tumors. To determine which genes were regulated synergistically by the inverse expression of NFkappaB and JNK, a pathway specific microarray analysis was performed. While either inhibition of NFkappaB or activation of JNK alone was unable to affect the IGFBP6 gene expression in microarray analysis, concomitant increase in JNK activation in the presence of NFkappaB inhibition increased the expression of this gene significantly. Synergistic increase in IGFBP6 gene expression was also confirmed by RT-PCR and Northern blot analysis of transfected cells. Accordingly, the levels of IGFBP6 protein secretion rose synergistically when JNK was over-expressed in NFkappaB knock down cells. In addition, increased expression of JNK in the absence of NFkappaB resulted in a significant induction of cell death in oral tumors when either left untreated or treated with TNF-alpha and TPA. Moreover, when JNK was inhibited by dominant negative JNK (APF), a significant decrease in cell death could be observed in TNF-alpha and TPA treated NFkappaB knock down oral tumors. Therefore, increased induction of IGFBP6 gene or protein expression in oral tumors could be regarded as a potential predictive marker of tumor sensitivity and could be used for prognostic purposes, since a significant correlation could be observed between increased induction of apoptotic cell death and elevated levels of IGFBP6 in these tumors.

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage.

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.

Mechanisms of N-acetyl cysteine-mediated protection from 2-hydroxyethyl methacrylate-induced apoptosis.

Resin-based materials are now commonly used in dentistry in restorative materials as well as in endodontic sealers. These materials have been shown to be cytotoxic. The mechanisms by which resin-based materials mediate their adverse effects have not been completely elucidated. Here we show that 2-hydroxyethyl methacrylate (HEMA) induces apoptotic cell death in oral keratinocytes and immune cells through the intrinsic cell death pathway. Functional loss and cell death induced by HEMA was significantly inhibited in the presence of N-acetyl cysteine (NAC) treatment. In addition, HEMA induced a decrease in mitochondrial membrane potential, and an increase in cleaved caspases was potently inhibited in the presence of NAC treatment. Overall, the results reported in this article indicate that NAC is an effective chemoprotectant that can safely be used to protect the pulp and the surrounding tissues from adverse effects of dental restorative and endodontic materials.

Epidermal growth factor receptor vIII expression in U87 glioblastoma cells alters their proteasome composition, function, and response to irradiation.

Little is known about the factors that influence the proteasome structures in cells and their activity, although this could be highly relevant to cancer therapy. We have previously shown that, within minutes, irradiation inhibits substrate degradation by the 26S proteasome in most cell types. Here, we report an exception in U87 glioblastoma cells transduced to express the epidermal growth factor receptor vIII (EGFRvIII) mutant (U87EGFRvIII), which does not respond to irradiation with 26S proteasome inhibition. This was assessed using either a fluorogenic substrate or a reporter gene, the ornithine decarboxylase degron fused to ZsGreen (cODCZsGreen), which targets the protein to the 26S proteasome. To elucidate whether this was due to alterations in proteasome composition, we used quantitative reverse transcription-PCR to quantify the constitutive (X, Y, Z) and inducible 20S subunits (Lmp7, Lmp2, Mecl1), and 11S (PA28alpha and beta) and 19S components (PSMC1 and PSMD4). U87 and U87EGFRvIII significantly differed in expression of proteasome subunits, and in particular immunosubunits. Interestingly, 2 Gy irradiation of U87 increased subunit expression levels by 16% to 324% at 6 hours, with a coincident 30% decrease in levels of the proteasome substrate c-myc, whereas they changed little in U87EGFRvIII. Responses similar to 2 Gy were seen in U87 treated with a proteasome inhibitor, NPI0052, suggesting that proteasome inhibition induced replacement of subunits independent of the means of inhibition. Our data clearly indicate that the composition and function of the 26S proteasome can be changed by expression of the EGFRvIII. How this relates to the increased radioresistance associated with this cell line remains to be established.

Potential contribution of naïve immune effectors to oral tumor resistance: role in synergistic induction of VEGF, IL-6, and IL-8 secretion.

The aim of this study is to identify the phenotype of resistant oral tumors, and to delineate the contribution of immune effectors to resistance of oral tumors. UCLA-1 oral tumors which were resistant to NK cell mediated cytotoxicity secreted increased amounts of IL-6, IL-1beta, GM-CSF, and IL-8 when cultured with or without immune effectors. In addition, the levels of vascular endothelial growth factor (VEGF) secretion in the co-cultures of naïve immune effectors with UCLA-1 rose significantly when compared to tumor cells alone. IL-2 activated NK cells decreased VEGF secretion in all tumor cells. However, NK cells which were induced to undergo cell death with anti-CD16 antibody were not only unable to decrease VEGF secretion, but they also contributed further to the increase in VEGF secretion by oral tumors. Overall, we show in this paper that naïve as well as non-viable immune effectors may contribute to the growth and resistance of oral tumors by triggering the secretion of key tumor cell growth factors.