Reactive oxygen species-dependent mechanisms of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in human carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer, and neuroblastoma. 4HPR induces apoptosis in various cancer cells and production of reactive oxygen species (ROS) has been suggested as a possible cause underlying these effects. However, the mechanisms governing these effects by 4HPR are not fully elucidated. In this study, we explored the mechanisms of 4HPR-induced ROS increase and apoptosis in human cancer cells. ^ First, we identified genes modulated by 4HPR using oligonucleotide gene expression arrays and found that they fall into specific functional canonical pathways and gene networks using Ingenuity Pathways Analysis®. Further analysis has shown that 4HPR induced up-regulation of Endoplasmic Reticulum (ER)-related genes such as Heat shock proteins 70 and 90 and the transcriptional factor, GADD153. These findings were validated using quantitative real-time PCR. ^ Second, we found that 4HPR induced extensive ER stress evidenced by dilation of the ER and endoribonuclease-mediated splicing and activation of the transcriptional factor, XBP-1. In addition, 4HPR induced the up-regulation of various ER stress-related genes and their protein products, as well as cleavage and activation of the ER specific Caspase-4. Concomitantly with XBP-1 splicing, all of these effects were dependent on ROS generation by 4HPR. Furthermore, chemical inhibition and RNA interference studies revealed a novel pro-apoptotic role for HSP70/A1A in 4HPR-mediated apoptosis. ^ Third, we observed rapid activation of the small GTPase Rac by 4HPR which was upstream of ROS generation. Inhibition of Rac activity or silencing of its expression by RNA interference inhibited ROS generation and apoptosis induction by 4HPR. siRNA targeting PAK1 and expression of a dominant negative Rac, decreased 4HPR-mediated ROS generation, while expression of a constitutive active Rac increased basal and 4HPR-induced ROS generation and PARP cleavage. Furthermore, metastatic cancer cells exhibited higher Rac activation, ROS generation, and cell growth inhibition due to 4HPR exposure compared to their primary cancer cell counterparts. ^ These findings provide novel insights into 4HPR-mediated ROS generation and apoptosis induction and support the use of ROS inducing agents such as 4HPR against metastatic cancer cells. ^

cAMP regulates IL-2 receptor signaling in human T cells

Proper immune system function is dependent on positive and negative regulation of T cell signaling pathways. Full T cell activation requires sequential signaling through the T cell receptor (TCR), costimulatory molecules and the IL-2 receptor (IL-2R). The IL-2R associated Janus tyrosine kinase 3 (Jak3), as well as Signal transducer and activator of transcription 5 (Stat5), are required for normal T cell function and survival. Constitutive activation of Jak3 and Stat5 have been linked to cancers of hematopoietic origin, including certain lymphomas and leukemias. ^ The production of cAMP by adenylate cyclase has been shown to negatively regulate human TCR mediated cell proliferation. Since cAMP has been shown to negatively regulate T cell activation, we sought to investigate whether crosstalk exists between cAMP and IL-2R signaling. The first objective of this study was to determine the effect of cAMP on the activation of IL-2R signaling molecules Jak3 and Stat5. We found that the potent adenylate cyclase activator, forskolin, inhibited IL-2 activation of Jak3 and Stat5. Indeed, in vitro kinase assays and electrophoretic mobility shift assays verified a loss of Jak3 enzymatic activity and Stat5 DNA binding ability, respectively. Further analysis of IL-2R signaling showed that forskolin treatment reduced IL-2 induced association of the IL-2Rβ and γc chain. ^ Because cAMP activates protein kinase A (PKA), the second objective was to determine the role for PKA in the cAMP directed regulation of IL-2R signaling intermediates. Interestingly, forskolin induced serine phosphorylation of Jak3, suggesting that cAMP can directly regulate Jak3 via activation of a serine/threonine kinase. Indeed, phosphoamino acid analysis revealed that PKA was able to induce Jak3 serine phosphorylation in the human leukemia cell line MT-2. In addition, in vitro kinase assays established that PKA can directly inhibit Jak3 enzymatic activity. Collectively, these data indicate that cAMP negatively regulates IL-2R signaling via various effector molecules by a previously unrecognized mechanism. This new data suggests that the Jak3/Stat5 pathway may be regulated by various pharmacological agents that stimulate cAMP production and thus can be used to uncouple some types of T cell mediated diseases. ^

Survival analysis of circulating tumor cells in triple-negative breast cancer

Background. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths in 2008. [1] Triple-negative breast cancer (TNBC) is an aggressive phenotype comprising 10–20% of all breast cancers (BCs). [2-4] TNBCs show absence of estrogen, progesterone and HER2/neu receptors on the tumor cells. Because of the absence of these receptors, TNBCs are not candidates for targeted therapies. Circulating tumor cells (CTCs) are observed in blood of breast cancer patients even at early stages (Stage I & II) of the disease. Immunological and molecular analysis can be used to detect the presence of tumor cells in the blood (Circulating tumor cells; CTCs) of many breast cancer patients. These cells may explain relapses in early stage breast cancer patients even after adequate local control. CTC detection may be useful in identifying patients at risk for disease progression, and therapies targeting CTCs may improve outcome in patients harboring them. Methods . In this study we evaluated 80 patients with TNBC who are enrolled in a larger prospective study conducted at M D Anderson Cancer Center in order to determine whether the presence of circulating tumor cells is a significant prognostic factor in relapse free and overall survival . Patients with metastatic disease at the time of presentation were excluded from the study. CTCs were assessed using CellSearch System™ (Veridex, Raritan, NJ). CTCs were defined as nucleated cells lacking the presence of CD45 but expressing cytokeratins 8, 18 or 19. The distribution of patient and tumor characteristics was analyzed using chi square test and Fisher's exact test. Log rank test and Cox regression analysis was applied to establish the association of circulating tumor cells with relapse free and overall survival. Results. The median age of the study participants was 53years. The median duration of follow-up was 40 months. Eighty-eight percent (88%) of patients were newly diagnosed (without a previous history of breast cancer), and (60%) of patients were chemo naïve (had not received chemotherapy at the time of their blood draw for CTC analysis). Tumor characteristics such as stage (P=0.40), tumor size (P=69), sentinel nodal involvement (P=0.87), axillary lymph node involvement (P=0.13), adjuvant therapy (P=0.83), and high histological grade of tumor (P=0.26) did not predict the presence of CTCs. However, CTCs predicted worse relapse free survival (1 or more CTCs log rank P value = 0.04, at 2 or more CTCs P = 0.02 and at 3 or more CTCs P < 0.0001) and overall survival (at 1 or more CTCs log rank P value = 0.08, at 2 or more CTCs P = 0.01 and at 3 or more CTCs P = 0.0001. Conclusions. The number of circulating tumor cells predicted worse relapse free survival and overall survival in TNBC patients.^

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

The transcription factor nuclear factor kappa B (NFkappaB) maintains TRAIL resistance in human pancreatic cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF family of cytokines that induces apoptosis in a variety of tumor cells while sparing normal cells. However, many human cancer cell lines display resistance to TRAIL-induced apoptosis and the mechanisms contributing to resistance remain controversial. Previous studies have demonstrated that the dimeric transcription factor Nuclear Factor kappa B (NFκB) is constitutively active in a majority of human pancreatic cancer cell lines and primary tumors, and although its role in tumor progression remains unclear it has been suggested that NFκB contributes to TRAIL resistance. Based on this, I examined the effects of NFκB inhibitors on TRAIL sensitivity in a panel of nine pancreatic cancer cell lines. I show here that inhibitors of NFκB, including two inhibitors of the proteasome (bortezomib (Velcade™, PS-341) and NPI-0052), a small molecule inhibitor of IKK (PS1145), and a novel synthetic diterpene NIK inhibitor (NPI-1342) reverse TRAIL resistance in pancreatic cancer cell lines. Further analysis revealed that the expression of the anti-apoptosic proteins BclXL and XIAP was significantly decreased following exposure to these inhibitors alone and in combination with TRAIL. Additionally, treatment with NPI0052 and TRAIL significantly reduced tumor burden relative to the control tumors in an L3.6pl orthotopic pancreatic xenograft model. This was associated with a significant decrease in proliferation and an increase in caspase 3 and 8 cleavage. Combination therapy employing PS1145 or NPI-1342 in combination with TRAIL also resulted in a significant reduction in tumor burden compared to either agent alone in a Panc1 orthotopic xenograft model. My studies show that combination therapy with inhibitors of NFκB alone and TRAIL is effective in pre-clinical models of pancreatic cancer and suggests that the approach should be evaluated in patients. ^

Regulation of squamous cell differentiation in human head and neck carcinoma cells by retinoids

Retinoids have been found to be effective in the prevention of premalignant lesions and second primary cancers in the upper aerodigestive tract. Further development of retinoids for prevention and therapy of head and neck squamous cell carcinoma (HNSCC) requires a better understanding of their mechanism of action on the growth and differentiation of such cells. I have chosen to employ cultured HNSCC cell lines as a model system for investigating the mechanism underlying the effects of retinoids. These cells are useful because all-trans retinoic acid (ATRA) inhibits their proliferation. Furthermore, two HNSCC cell lines were found to express three squamous differentiation (SqD) markers characteristic of normal keratinocytes and ATRA suppressed the expression of these markers as reported for normal keratinocytes. It is thought that nuclear retinoic acid receptors (RARs and RXRs), which act as DNA-binding transcription modulating factors, mediate the effects of retinoids on the growth and differentiation of normal and tumor cells. I found that all four cell lines examined expressed RAR-$\alpha ,$ RAR-$\tau ,$ and RXR-$\alpha$ and three of four expressed RAR-$\beta .$ ATRA treatment increased the level of RAR-$\alpha ,$ -$\beta ,$ and -$\tau$ in four cell lines. Two HNSCC cell lines that exhibited a progressive increase in the expression of SqD markers during growth in culture also showed a concurrent decrease in RAR-$\beta$ level. Moreover, increasing concentrations of RA suppressed the SqD marker while inducing RAR-$\beta$ mRNA. Several synthetic retinoids which exhibit a preference for binding to specific nuclear RARs showed a differential ability to inhibit cell proliferation, transactivate transcription of the reporter genes (CAT and luciferase) from the RA response element (RARE) of the RAR-$\beta$ gene, and induce RAR-$\beta$ expression. Those retinoids that were effective inducers of RAR-$\beta$ also suppressed SqD effectively, indicating an inverse relationship exists between the expression of RAR-$\beta$ and SqD. This inverse relationship suggests a role for RAR-$\beta$ in the suppression of SqD. ^

Dissection of antitumor activity by lymphokine -activated killer cells: Role of FcR+ precursors and obligatory role of tumor necrosis factor-$\alpha$ in antibody -dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) cultured for varying lengths of time in IL-2 are able to mediate antibody independent cellular cytotoxicity (AICC) as well as antibody dependent cellular cytotoxicity (ADCC) against a wide range of tumor targets. The objective of our study is to determine the cytotoxic potential of the subset of LAK cells involved in ADCC, the tumor recognition mechanism in ADCC, the kinetics of ADCC mediated by PBL cultured under various conditions and the role of TNF-$\alpha$ in the development and maturation of ADCC effectors in the LAK population.^ The model system in this study for ADCC used a monoclonal antibody 14G2a (IgG2a), that recognizes the GD2 epitope on human melanoma cell line, SK-Mel-1. The target recognition mechanism operative in AICC (traditionally known as lymphokine activated killing or LAK) is an acquired property of these IL-2 activated cells which confers on them the unique ability to distinguish between tumor and normal cells. This recognition probably involves the presence of a trypsin sensitive N-linked glycoprotein epitope on tumor cells. Proteolytic treatment of the tumor cells with trypsin renders them resistant to AICC by PBL cultured in IL-2. However, ADCC is unaffected. This ADCC, mediated by the relatively small population of cells that are positive for the Fc receptor for IgG (FcR), is an indication that this subset of "LAK" cells does not require the trypsin sensitive epitope on tumor cells to mediate killing. Enriching PBL for FcR+ cells markedly enhanced both AICC and ADCC and also reduced the IL-2 requirement of these cells.^ The stoichiometry of Fc receptor (FcR) expression on the cytotoxic effectors does not correlate with ADCC lytic activity. Although FcRs are necessary to mediate ADCC, other factors, appear to regulate the magnitude of cytolytic activity. In order to investigate these putative factors, the kinetics of ADCC development was studied under various conditions (in IL-2 (10u/ml) and 100u/ml), in IL-2(10u/ml) + TNF$\alpha$ (500u/ml) and in TNF-$\alpha$ (500u/ml) alone). Addition of exogenous TNF-$\alpha$ into the four hour cytotoxicity assay did not increase ADCC, nor did anti-TNF antibodies result in inhibition. On the other hand, addition of anti-TNF antibodies to PBL and IL-2 for 24 hours, resulted in a marked inhibition of the ADCC, suggesting that endogenous TNF-$\alpha$ is obligatory for the maturation and differentiation of ADCC effectors. ^

Role and regulation of c-KIT gene expression in tumor growth and metastasis of human melanoma

The purpose of this study was to investigate the role of the c-KIT receptor in the progression of human melanoma and the mechanism(s) for the regulation of c-KIT gene expression in human melanoma.^ The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) (metastatic phenotype) are not well-defined. Expression of the tyrosine-kinase receptor c-KIT progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that the metastasis of human melanoma is associated with the loss of c-KIT expression, highly metastatic A375SM cells, which express very low or undetectable levels of c-KIT, were tranduced with the human c-KIT gene. We demonstrated that enforced c-KIT expression in highly metastatic human melanoma cells significantly suppressed their tumorigenicity and metastatic propensity in nude mice. In addition, we showed that the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis.^ Furthermore, we investigated the possible mechanism(s) for the down-regulation of c-KIT gene expression in malignant melanoma. Sequence analysis of the c-KIT promoter indicated that this promoter contains several consensus binding-site sequences including three putative AP2 and two Myb sites. Although Myb was shown to be associated with c-KIT expression in human hemotopoietic cells, we found no correlation between c-KIT expression and Myb expression in human melanoma cell lines. In contrast, we showed that c-KIT expression directly correlates with expression of AP2 in human melanoma cells. We found that highly metastatic cells do not express the transcription factor AP2. Expression of AP2 in A375SM cells (c-KIT-negative and AP2-negative) was enough to restore luciferase activity driven by the c-KIT promoter in a dose-dependent manner. On the other hand, co-expression of the dominant-negative form of AP2 (AP2B) in Mel-501 cells (c-KIT-positive and AP2-positive) resulted in two-fold reduction in luciferase activity. Electrophoretic mobility shift assays revealed that the c-KIT promoter contains functional AP2 binding sites which could associate with AP2 protein. Endogenous c-KIT gene expression levels were elevated in AP2 stably-transfected human melanoma A375SM cells. Expression of exogenous AP2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. The c-KIT ligand, SCF, also induced apoptosis in the AP2 stably-transfected A375SM cells. The identification of AP2 as an important regulator for c-KIT expression suggests that AP2 may have tumor growth and metastasis inhibitory properties, possibly mediated through c-KIT/SCF effects on apoptosis of human melanoma cells. Since AP2 binding sites were found in the promoters of other genes involved in the progression of human melanoma, such as MMP2 (72 kDa collagenase), MCAM/MUC18 and P21/WAF-1, our findings suggest that loss of AP2 expression might be a crucial event in the development of malignant melanoma. ^

IL -12 inhibits the metastatic potential of osteosarcoma cells and induces tumor regression via a mechanism involving Fas /Fas ligand pathway

Despite multiple changes in the adjuvant chemotherapy regimens used to treat osteosarcoma (OS), the 2-year metastasis-free survival has remained at 65–70% for the past 10 years. Characterizing the molecular determinants that permit metastatic spread of tumor cells is a crucial element in developing new approaches for the treatment of osteosarcoma. Since OS metastasizes almost exclusively to the lung, an organ with constitutive Fas ligand (FasL) expression, we hypothesized that the expression of Fas (CD95, APO-1) by OS cells may play a role in the ability of these cells to form lung metastases. Fas expression was quantified in human SAOS-2 OS cells and selected variants (LM2, LM4, LM5, LM6, LM7). Using northern blot, FACS and RT-PCR analysis, low Fas expression was found to correlate with higher metastatic potential in these cell lines. The highly metastatic LM7 cell line was transfected with the full-length human Fas gene and injected into athymic nude mice. The median number of metastatic nodules per mouse fell from over 200 to 1.1 and the size of the nodules decreased from a range of 0.5–9.0 mm to less than 0.5 mm in the Fas-transfected cell line compared to the native LM7 cell line. Additionally, the subsequent incidence of lung metastases was lower in the Fas-expressing cell line. IL-12 was seen to upregulate Fas expression in the highly metastatic LM sublines in vitro. To visualize the effects of IL-12 in vivo, nude mice were injected with LM7 cells and treated biweekly for 4 weeks with Ad.mIL-12, saline control or Ad.βgal. Lung sections were analyzed via immunchistochemistry for Fas expression. A higher expression of Fas was found in tumors from mice receiving IL-12. To study the mechanism by which IL-12 upregulates Fas, LM7 cells were transfected with a luciferase reporter gene construct containing the full-length human fas promoter. Treatment with IL-12 increased luciferase activity. We therefore conclude that IL-12 influences the metastatic potential of OS cells by upregulating the fas promoter, resulting in increased cell surface Fas expression and susceptibility to Fas-induced cell death. ^

Activation of matrix metalloproteinase -9 (MMP-9) by heregulin in human breast cancer cells involves multiple signaling pathways

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor invasion and angiogenesis. Secretion of MMP-9 has been reported in various cancer types including lung cancer, brain cancer, colon cancer, and breast cancer. Heregulin is a growth factor that regulates growth and differentiation of normal breast cells as well as mammary tumor cells. To study the role of heregulin in breast cancer metastasis, we tested whether heregulin may regulate MMP-9 secretion. By screening a panel of breast cancer cell line for their ability to respond to heregulin and produce MMP-9, we have found that MMP-9 secretion can be induced by heregulin-β1 in two breast cancer cell lines, SKBr3 and MCF-7. In both cell lines, increase of MMP-9 activity as shown by zymography was accompanied by increased protein level as well as mRNA level of MMP-9. Using a reporter luciferase assay, we have identified that proximal −670bp promoter of MMP-9 had similar activity to a 2.2kb MMP-9 promoter in response to heregulin stimulation. Heregulin treatment of SKBr3 and MCF-7 activated multiple signaling pathways inside cells. These include the Erk pathway, the p38 kinase pathway, PKC pathway, and PI-3K pathway. To examine which pathways are involved in MMP-9 activation by heregulin, we have used a panel of chemical inhibitors to specifically inhibit each one of these pathways. Ro-31-8220 (PKC inhibitor) and SB203580 (p38 kinase inhibitor) completely blocked heregulin activation of MMP-9. On the other hand, PD098059 (MEK-1 inhibitor) partially blocked MMP-9 activation, whereas PI-3K inhibitor, wortmannin, had no effect. Therefore, at least three signaling pathways are involved in activation of MMP-9 by heregulin. Since MMP-9 is tightly associated with metastatic potential, our study also suggests that heregulin may enhance breast tumor metastasis through induction of MMP-9 expression. ^

Dual Targeting of Tumor Angiogenesis and Chemotherapy by Endostatin- Cytosine Deaminase-Uracil PhosphoribosylTransferase

Antiangiogenesis is a promising anti-tumor strategy through inhibition tumor vascularformation to suppress tumor growth. Targeting specific VEGF/R has been showntherapeutic benefits in many cancer types and become a first approvedantiangiogenic modalities by Food and Drug Administration (FDA) in United States.However, interruption of homeostasis in normal tissues that is likely due to theinhibition of VEGF/R signaling pathway induces unfavorable side effects. Moreover,cytostatic nature of antiangiogenic drugs frequently causes less tumor cell specifickilling activity, and cancer cells escaped from cell death induced by these drugseven gain more malignant phenotypes, resulting in tumor invasion and metastasis.To overcome these issues, we developed a novel anti-tumor therapeutic EndoCDfusion protein which linked endostatin (Endo) to cytosine deaminase-uracilvphosphoribosyl transferase (CD). Endo targets unique tumor endothelial cells toprovide tumor-specific antiangiogenesis activity and also carries CD to the localtumor area, where it serves nontoxic prodrug 5-fluorocytosine (5-FC) enzymaticconversion reaction to anti-metabolite chemotherapy drug 5-fluorouracil (5-FU). Wedemonstrated that 5-FU concentration was highly increased in tumor sites, resultingin high level of endothelial cells and tumor cells cytotoxic efficacy. Furthermore,EndoCD/5-FC therapy decreased tumor growth and colorectal liver metastasisincident compared with bevacizumab/5-FU treatment in human breast and colorectalliver metastasis orthotropic animal models. In cardiotoxicity safety profile,EndoCD/5-FC is a contrast to bevacizumab/5-FU; lower risk of cardiotoxicityinduction or heart function failure was found in EndoCD/5-FC treatment thanbevacizumab/5-FU does in mice. EndoCD/5-FC showed more potent therapeuticefficacy with high safety profile and provided stronger tumor invasion or metastasisinhibition than antiangiogenic drugs. Together, EndoCD fusion protein with 5-FCshowed dual tumor targeting activities including antiangiogenesis and tumor localchemotherapy, and it could serve as an alternative option for antiangiogenic therapy.

Radiolabeled human monoclonal IgM for intracompartmental cancer therapy

Radioimmunotherapy (RIT) with i.v. administered radiolabeled IgG can selectively irradiate tumor cells in vivo. However, it only provides effective therapy for lymphomas. Intracompartmental RIT with radiolabeled human monoclonal IgM may allow curative treatment of solid tumors by increasing tumor deposition of radioactivity, reducing systemic toxicity and allowing repeated administration. This hypothesis was tested in nude mouse models with IgM radiolabeled with indium-111 $\rm(\sp{111}In)$ or yttrium-90 $\rm(\sp{90}Y).$ The use of two radioisotopes, $\rm\sp{111}In$ for imaging and $\rm\sp{90}Y$ for therapy, allow for more quantitative and cautious development of RIT.^ Radiolabled 2B12, an IgM reactive with human ovarian carcinomas was tested by i.v. and intraperitoneal (i.p.) administration in nude mice bearing i.p. nodules of a human ovarian carcinoma cell line (SKOV3 NMP2). Radiolabeled CR4E8, an IgM reactive with human squamous cell carcinomas was tested by i.v. and intralesional (i.l.) administration in nude mice bearing subcutaneous tumors of a human head and neck squamous cell carcinoma cell line (886). These two models were selected to test proof of concept. Radiolabeled irrelevant IgM (CH-1B9), and $\rm\sp{90}Y$-aggregate served as specificity controls. Biodistribution was performed by excising, weighing and then measuring the radioactivity of tumor and normal organs. Therapy was conducted with i.p. $\rm\sp{90}Y$-labeled 2B12 using both single and fractionated administration and with i.l. $\rm\sp{90}Y$-labeled CR4E8 using single administration. Mice were monitored for tumor response, survival and systemic toxicity.^ Intracompartmental administration of radiolabeled IgM produced immediate high and prolonged tumor deposition of radioactivity with low normal tissue uptake. In contrast, i.v. administration resulted in low tumor, but high liver and spleen uptake. Similar biodistributions were demonstrated for $\rm\sp{111}In$- and $\rm\sp{90}Y$-labeled IgM. Intraperitoneal therapy with $\rm\sp{90}Y$-labeled 2B12 increased survival by approximately 12 days for every 100 $\rm\mu Ci$ of activity without significant toxicity for single (0-300 $\rm\mu Ci)$ and fractionated (150-510 $\rm\mu Ci)$ administration. Intralesional therapy with $\rm\sp{90}Y$-labeled CR4E8 (150-400 $\rm\mu Ci)$ induced prolonged complete regressions. Significant local or systemic toxicity was not observed.^ Intracompartmental RIT with radiolabeled tumor-reactive human monoclonal IgM can selectively irradiate tumor cells. Intracompartmental radiolabled IgM can significantly extend the survival of treated mice with minimal toxicity. It deserves further development as a new cancer therapy. ^

THE ROLE OF B CELLS IN THE T CELL DEPENDENT RELEASE OF HUMAN B CELL GROWTH FACTOR

Quiescent human B cells are postulated to go through activation and proliferation phases before undergoing differentiative phase for immunoglobulin secretion. The present studies address some of the aspects of activation and proliferation phase of normal human B cells. The definitions of signals responsible for B cell activation and proliferation resulted in the development of a highly specific, reproducible B cell growth factor (BCGF) assay. This BCGF bioassay utilizes activation by rabbit anti-human IgM-antibody. The functional specificity of this assay for measuring BCGF activity was demonstrated by the finding that target B cells proliferated but did not differentiate. The factor specificity was determined by specific absorption of BCGF by anti-IgM activated B cells. This assay was utilized for the studies of T-B cell collaboration and the essential function of monocytes in the production and/or release of B cell growth factor in a syngeneic in vitro system. It is apparent that highly purified T cells are poor producers of BCGF by themselves and require monocytes to secrete significant quantities of BCGF upon PHA stimulation. Macrophage soluble factor, Interleukin 1, is capable of replacing monocyte function for the release of BCGF by activated T cells. In our studies, B cells are incapable to function as accessory cells to replace monocyte function. Normal B cells are also not capable of producing BCGF under our experimental observations. However, the addition of these B cells at an optimum cell density (T:B ratio 1:1) doubles the monocyte dependent release of BCGF by syngeneic T cells. The augmentative role of B cells is expanded to understand the mechanism of BCGF release by T cells. It is observed from our studies that DR antigen of B cell surface is involved in the release of BCGF. The functional difference between DR of B cells and monocytes is observed as IL-1 could replace DR-treated monocytes whereas failed to replace DR-treated B cells for the release of BCGF by T cells. This functional difference may be attributed to the reported microheterogeneity in DR of B cells and monocytes. The addition of irradiated B cells increased the monocyte dependent T cell proliferation, suggesting the increase of T cell pool for BCGF release. In summary, the development of a biological assay specific for B cell growth factor led to the delineation of an interesting role of B cells in the release of its own growth factor by T cells. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

NFkappaB and STAT binding elements participate in regulation of MUC1 expression in normal and transformed mammary epithelial cells

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in several cancers of epithelial origin, including those of breast, pancreas, lung, ovary, and colon. Functions of MUC1 include protection of mucosal epithelium, modulation of cellular adhesion, and signal transduction. Aberrantly increased expression of MUC1 in cancer cells promotes tumor progression through adaptation of these functions. Some regulatory elements participating in MUC1 transcription have been described, but the mechanisms responsible for overexpression are largely unknown. A region of MUC1 5′ flanking sequence containing two conserved potential cytokine response elements, an NFκB site at −589/−580 and a STAT binding element (SBE) at −503/−495, has been implicated in high level expression in breast and pancreatic cancer cell lines. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. ^ T47D breast cancer cells and normal human mammary epithelial cells (HMEC) were used to determine the roles of the κB site and SBE in basal and stimulated expression of MUC1. Treatment of T47D cells and HMEC with interferon-γ (IFNγ) alone enhanced MUC1 expression at the level of transcription, and the effect of IFNγ was further stimulated by tumor necrosis factor-α (TNFα). MUC1 responsiveness to these cytokines was modest in T47D cells but clearly evident in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that the κB site at −589/−580 and the SBE at −503/−495 and were required for cooperative stimulation by TNFα and IFNγ. Electrophoretic mobility shift assays (EMSA) revealed that the synergy was mediated not by cooperative binding of transcription factors but by the independent actions of STAT1α and NFκB p65 on their respective binding sites. Independent mutations in the κB site and SBE abrogated cytokine responsiveness and reduced basal MUC1 promoter activity by 45–50%. However, only the κB site appeared to be constitutively activated in T47D cells, in part by NFκB p65. These findings implicate two cytokine response elements in the 5 ′ flanking region of MUC1, specifically a κB site and a STAT binding element, in overexpression of MUC1 in breast cancer cells. ^