TFP5, a Peptide Inhibitor of Aberrant and Hyperactive Cdk5/p25, Attenuates Pathological Phenotypes and Restores Synaptic Function in CK-p25Tg Mice.

It has been reported that cyclin-dependent kinase 5 (cdk5), a critical neuronal kinase, is hyperactivated in Alzheimer's disease (AD) and may be, in part, responsible for the hallmark pathology of amyloid plaques and neurofibrillary tangles (NFTs). It has been proposed by several laboratories that hyperactive cdk5 results from the overexpression of p25 (a truncated fragment of p35, the normal cdk5 regulator), which, when complexed to cdk5, induces hyperactivity, hyperphosphorylated tau/NFTs, amyloid-ß plaques, and neuronal death. It has previously been shown that intraperitoneal (i.p.) injections of a modified truncated 24-aa peptide (TFP5), derived from the cdk5 activator p35, penetrated the blood-brain barrier and significantly rescued AD-like pathology in 5XFAD model mice. The principal pathology in the 5XFAD mutant, however, is extensive amyloid plaques; hence, as a proof of concept, we believe it is essential to demonstrate the peptide's efficacy in a mouse model expressing high levels of p25, such as the inducible CK-p25Tg model mouse that overexpresses p25 in CamKII positive neurons. Using a modified TFP5 treatment, here we show that peptide i.p. injections in these mice decrease cdk5 hyperactivity, tau, neurofilament-M/H hyperphosphorylation, and restore synaptic function and behavior (i.e., spatial working memory, motor deficit using Rota-rod). It is noteworthy that TFP5 does not inhibit endogenous cdk5/p35 activity, nor other cdks in vivo suggesting it might have no toxic side effects, and may serve as an excellent therapeutic candidate for neurodegenerative disorders expressing abnormally high brain levels of p25 and hyperactive cdk5.

Educational Needs Assessment Highlights Several Areas of Emphasis in Teaching Evidence-Based Medicine Skills to Physician Assistant Students.

PURPOSE: An assessment of educational needs is essential for curricular reform in medical education. Using the conceptual framework of needs assessment, this study aimed to determine which content should be emphasized in teaching evidence-based medicine (EBM) skills to physician assistant (PA) students. METHODS: Key content areas were identified from the published literature and objectives for previous courses. A questionnaire-type needs assessment instrument was created and given to a graduating class of PA students (n = 21) at the University of North Dakota. The response format had two 5-option scales, one to assess current skill levels and the other to assess ideal skill levels. Means for each category were calculated, and a mean difference analysis was performed. RESULTS: An average mean difference of 0.5 was noted in 3 domains (information retrieval skills, writing skills, and overall gains), and a mean difference of 0.7 was noted in one domain (statistical skills). Items with a mean difference of ≥ 0.7 were identified for prioritization for curricular reform. Open-ended input from respondents substantiated the need for greater emphasis on these content areas. CONCLUSION: Several content areas related to EBM skills can be identified and prioritized through a systematically conducted educational needs assessment. This method can be used to identify discrepancies between the existing and ideal states of affairs in PA education.

Lipoprotein abnormalities in patients with type 2 diabetes and metabolic syndrome.

Cardiovascular disease remains the leading cause of death in men and women in the United States. Aggressive treatment of insulin resistance and its associated lipid abnormalities remains a top priority for preventing cardiovascular morbidity and mortality.

Treatment of restless legs syndrome with iron infusion therapy.

Initially, the patient in this case received CPAP for sleep apnea and pramipexole for RLS. Her symptoms failed to resolve, which led to consultation with a sleep specialist who recommended diagnostic studies that identified an underlying iron/ferritin deficiency. After receiving IV iron therapy, the patient's RLS symptoms resolved. Only about one-quarter of patients with RLS symptoms receive an appropriate diagnosis, and thus, many are treated less than optimally. Agents used to treat RLS have been heavily advertised, and patients may request a prescription from their health care provider. This case reaffirms the importance of the medical history and diagnostic studies when evaluating a patient with symptoms of RLS. Screening first for iron/ferritin deficiency may prevent subjecting patients to the potential side effects of unnecessary medications.

Development and evaluation of BioScore: a biomarker panel to enhance prognostic algorithms for clear cell renal cell carcinoma.

BACKGROUND: The authors previously showed that increased tumor expression levels of B7-H1, survivin, and Ki-67 are independent predictors of poor outcome for patients with clear cell renal cell carcinoma (ccRCC). In the current study, they described the creation of a scoring system based on this panel of biomarkers that can be used in tandem with existing clinicopathologic features and algorithms to improve ccRCC outcome prediction. METHODS: The authors used immunohistochemistry to determine tumor expression levels of B7-H1, survivin, and Ki-67 for 634 consecutive ccRCC patients. A multivariate model verified that each biomarker was independently associated with RCC-specific death after adjusting for the remaining 2. A biomarker-based panel, termed BioScore, was generated to predict the likelihood of RCC-specific death. BioScore was tested for its ability to enhance the performance of several clinicopathologic features and algorithms. RESULTS: Patients with high BioScores were 5 times more likely to die from RCC compared with patients with low BioScores (hazard ratio, 5.03; 95% confidence interval, 3.82-6.61; P < .001). Multivariate adjustment for individual clinicopathologic features or existing prognostic algorithms failed to attenuate this positive association. Moreover, an examination of concordance indexes revealed that BioScore significantly enhanced the prognostic ability of each of the individual prognostic features or algorithms studied. CONCLUSIONS: The authors described the creation of BioScore, a biomarker-based scoring system that can be used in tandem with established prognostic algorithms to further enhance ccRCC outcome prediction. The need for external validation notwithstanding, they envision that BioScore can be readily updated as new biomarkers are identified.

T-cell coregulatory molecule expression in urothelial cell carcinoma: clinicopathologic correlations and association with survival.

PURPOSE: Aberrant expression of T-cell coregulatory molecules has been investigated as a mechanism by which certain cancers may evade host immune surveillance. We evaluated expression of the T-cell coregulators B7-H1, B7-H3, and PD-1 in urothelial cell carcinoma (UCC) of the bladder. EXPERIMENTAL DESIGN: Immunohistochemistry for B7-H1, B7-H3, and PD-1 was done on paraffin-embedded sections from 318 consecutive patients with UCC who underwent radical cystectomy. Expression was correlated with clinicopathologic outcomes and postoperative survival. RESULTS: B7-H3 was widely expressed in UCC, as 222 of 314 (70.7%) tumors showed positive staining. Expression of B7-H3 in UCC was significantly increased compared with adjacent, nontumor urothelium, as a median of 70% of tumor cells expressed B7-H3, compared with 20% of cells in nontumor specimens (P < 0.001). The increase in B7-H3 expression was independent of tumor stage (P = 0.13). Expression of B7-H1 by UCC tumors (P < 0.001) and PD-1 by tumor-infiltrating lymphocytes (P = 0.012) were significantly associated with increased pathologic stage. Patients who had received intravesical bacillus Calmette-Guerin before cystectomy tended to show increased expression of B7-H3 (P = 0.023) and PD-1 (P = 0.071) but were less likely to express B7-H1 (P = 0.027). Moreover, for the subset of patients with organ-confined disease (n = 167), B7-H1 expression independently predicted all-cause mortality after cystectomy (hazard ratio, 3.18; 95% confidence interval, 1.74-5.79; P < 0.001). CONCLUSIONS: B7-H3 is highly expressed in UCC across tumor stages, whereas B7-H1 and PD-1 expression are associated with advanced disease. B7-H1 expression predicts mortality after cystectomy for patients with organ-confined tumors. These molecules may represent novel diagnostic or prognostic markers, as well as therapeutic targets, for patients with UCC.

Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma.

PURPOSE: Although the prognostic value of B7-H1 and B7-H4 expression by tumor cells in clear cell renal cell carcinoma (ccRCC) has been established, the role of B7-H3 is unknown. As such, we evaluated the association of B7-H3 expression with clinicopathologic outcomes in patients treated for ccRCC. EXPERIMENTAL DESIGN: Nephrectomy specimens from 743 consecutive patients treated for ccRCC at our institution from 1990 to 1999 were evaluated for B7-H3 expression by immunohistochemical staining. Associations of B7-H3 expression with clinical and pathologic features were evaluated using chi2 and Fisher's exact tests. Associations of B7-H3 expression with death from RCC were evaluated using Cox proportional hazards regression models. RESULTS: B7-H3 expression by tumor cells or tumor vasculature was noted in 17% and 95% of specimens, respectively. The presence of either tumor cell or diffuse tumor vasculature expression of B7-H3 was present in 46% of specimens and was associated with multiple adverse clinical and pathologic features. After multivariable adjustment, the presence of either tumor cell or diffuse tumor vasculature B7-H3 expression was significantly associated with an increased risk of death from RCC (risk ratio, 1.38; 95% confidence interval, 1.03-1.84; P = 0.029). CONCLUSIONS: Both tumor cell and tumor vasculature B7-H3 expression convey important information to predict ccRCC outcomes. Collectively, our past and present studies pertaining to B7-H ligand expression indicate that ccRCC may use redundant mechanisms to compromise host antitumoral immunity. Future studies will focus on the effect of combined B7-H ligand expression in RCC.

Questionable relevance of gamma delta T lymphocytes in renal cell carcinoma.

Adoptive gammadelta T cell immunotherapy has moved briskly into clinical trials prompted by several small studies suggesting abundant accumulation of gammadelta T cells within renal cell carcinoma (RCC). In this study, we re-examined levels of gammadelta T cells within RCC tumors and correlated levels of these cells with pathologic features and outcome associated with this form of cancer. Tissues from 248 consecutive clear cell RCC tumors obtained from 2000 to 2003 were stained and quantified for total CD3+ and gammadelta T cells per mm2. Wilcoxon rank sum and Kruskal-Wallis tests were used to evaluate associations between T cell amounts and prognostic factors (age, gender, tumor size, stage, grade, tumor necrosis). Cox models were used to assess associations with RCC-specific death. Median numbers of total CD3+ and gammadelta T cells were 281/mm2 (interquartile range (IQR): 149-536) and 2.6/mm2 (IQR: 1.3-4.6), respectively. The median percentage of CD3+ T cells that were gammadelta T cells was 1.0% (IQR: 0.4-1.9). This low percentage of intratumoral gammadelta T cells was diluted even further with rising CD3+ T cell infiltration. Percentages of gammadelta T cells were not associated with even one single clinicopathologic feature examined. Median follow-up for this study was 3.1 years (48 patients died of RCC) and Cox analysis failed to demonstrate that gammadelta T cells (hazard ratio=1.02, p=0.25) were predictive of RCC-specific death. gammadelta T cells are rare and not recruited nor expanded within RCC tumors. Percentages of gammadelta T cells fail to correlate with any prognostic features of RCC nor specific death. As such, the role of gammadelta T cells in RCC immunobiology remains questionable.

Targeting molecular and cellular inhibitory mechanisms for improvement of antitumor memory responses reactivated by tumor cell vaccine.

Development of effective vaccination approaches to treat established tumors represents a focus of intensive research because such approaches offer the promise of enhancing immune system priming against tumor Ags via restimulation of pre-existing (memory) antitumoral helper and effector immune cells. However, inhibitory mechanisms, which function to limit the recall responses of tumor-specific immunity, remain poorly understood and interfere with therapies anticipated to induce protective immunity. The mouse renal cell carcinoma (RENCA) tumor model was used to investigate variables affecting vaccination outcomes. We demonstrate that although a whole cell irradiated tumor cell vaccine can trigger a functional antitumor memory response in the bone marrows of mice with established tumors, these responses do not culminate in the regression of established tumors. In addition, a CD103+ regulatory T (Treg) cell subset accumulates within the draining lymph nodes of tumor-bearing mice. We also show that B7-H1 (CD274, PD-L1), a negative costimulatory ligand, and CD4+ Treg cells collaborate to impair the recall responses of tumor-specific memory T cells. Specifically, mice bearing large established RENCA tumors were treated with tumor cell vaccination in combination with B7-H1 blockade and CD4+ T cell depletion (triple therapy treatment) and monitored for tumor growth and survival. Triple treatment therapy induced complete regression of large established RENCA tumors and raised long-lasting protective immunity. These results have implications for developing clinical antitumoral vaccination regimens in the setting in which tumors express elevated levels of B7-H1 in the presence of abundant Treg cells.

B7-H3 ligand expression by prostate cancer: a novel marker of prognosis and potential target for therapy.

B7 coregulatory ligands can be aberrantly expressed in human disease. In the context of cancer, these ligands may act as antigen-specific inhibitors of T-cell-mediated antitumoral immunity. We recently reported that B7-H1 expression by carcinomas of the kidney and bladder portends aggressive disease and diminished survival. The expression of these proteins in prostate cancer, however, has not been investigated. We evaluated B7-H3 and B7-H1 protein expression in the pathologic specimens of 338 men treated for clinically localized prostate cancer between 1995 and 1998 with radical retropubic prostatectomy. Expression levels of B7-H3 in prostate cancer were correlated with pathologic indicators of aggressive cancer as well as clinical outcome. We report that B7-H3 is uniformly and aberrantly expressed by adenocarcinomas of the prostate, high-grade prostatic intraepithelial neoplasia, and four prostate cancer cell lines, whereas B7-H1 is rarely expressed. B7-H3 is expressed by benign prostatic epithelia, although at a more reduced level relative to neoplastic tissue. Increasing levels of B7-H3 intensity correlate with worsening clinicopathologic features of prostate cancer. Marked B7-H3 intensity, present in 67 (19.8%) specimens, confers a >4-fold increased risk of cancer progression after surgery (risk ratio, 4.42; P < 0.001). A survey of normal tissues revealed that B7-H3 is expressed within the liver, urothelium, and fetal kidney. In summary, B7-H3 is aberrantly expressed in all prostate cancers and represents an independent predictor of cancer progression following surgery. Moreover, B7-H3 encompasses a novel diagnostic and potential therapeutic target for the clinical management of prostate cancer and, perhaps, other malignancies as well.

Tumor-infiltrating Foxp3-CD4+CD25+ T cells predict poor survival in renal cell carcinoma.

PURPOSE: Regulatory T cells (Tregs) have been implicated as inhibitors of antitumoral immunity, and evidence suggests that elimination of Tregs may augment natural and pharmacologic immunity. We tested for the presence of putative Tregs within renal cell carcinoma (RCC) tumors. EXPERIMENTAL DESIGN: We identified 170 patients who underwent radical or partial nephrectomy for clear cell RCC between 2000 and 2002. Specimens were stained with anti-CD4, anti-CD25, and anti-Foxp3 antibodies and examined using confocal microscopy. Associations of CD4(+)CD25(+)Foxp3(-) and CD4(+)CD25(+)Foxp3(+) T cells with death from RCC were evaluated using Cox proportional hazards regression models. RESULTS: At last follow-up, 46 of 170 patients had died; of these, 37 died from RCC at a median of 1.4 years following nephrectomy (range, 0-4.4). Among the 124 remaining patients, median follow-up was 3.7 years (range, 0-5.7). Forty-three (25.3%) tumors harbored CD4(+)CD25(+)Foxp3(+) T cells. The presence of Foxp3(+) T cells was not significantly associated with RCC death univariately. One hundred forty-three (84.1%) tumors harbored CD4(+)CD25(+)Foxp3(-) T cells. The indicator for >or=10% CD4(+)CD25(+)Foxp3(-) T cells was significantly associated with RCC death univariately [risk ratio (RR), 2.60; 95% confidence interval (95% CI), 1.35-4.98; P = 0.004], after adjusting for tumor B7-H1 expression (RR, 2.53; 95% CI, 1.32-4.85; P = 0.005) and lymphocytic infiltration (RR, 2.53; 95% CI, 1.32-4.87; P = 0.005). CONCLUSIONS: Increased presence of CD4(+)CD25(+)Foxp3(+) T cells was not significantly associated with RCC death. In contrast, CD4(+)CD25(+)Foxp3(-) T cells, which may represent a unique set of Tregs or activated helper T cells, was significantly associated with outcome.

Survivin and b7-h1 are collaborative predictors of survival and represent potential therapeutic targets for patients with renal cell carcinoma.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is an immunogenic tumor that can progress in the presence of an intact host immune system. We previously reported that survivin and B7-H1 are independently associated with disease progression and death when expressed by ccRCC tumors. Herein, we examine the clinical effect of ccRCC combined expression of both survivin and B7-H1. EXPERIMENTAL DESIGN: Specimens from 298 patients who underwent nephrectomy for ccRCC between 1990 and 1994 were immunohistochemically stained for survivin and B7-H1. Cancer-specific survival was estimated using the Kaplan-Meier method. Associations of both markers with ccRCC death were assessed using Cox proportional hazards regression models. RESULTS: At last follow-up, 94 patients died from ccRCC. Among the living patients, the median follow-up was 11.2 years (range, 0-15 years). There were 177 (59.4%) survivin(Low)/B7-H1(-), 51 (17.1%) survivin(Hi)/B7-H1(-), 29 (9.7%) survivin(Low)/B7-H1(+), and 41 (13.8%) survivin(Hi)/B7-H1(+) tumors. The 5-year cancer-specific survival rates for patients within each group were 89.3%, 59.7%, 70.0%, and 16.2%, respectively. Combined survivin(Hi)/B7-H1(+) expression was associated with ccRCC death univariately (risk ratio, 12.82; 95% confidence interval, 7.50-21.92; P < 0.001) and in multivariate analysis (risk ratio, 2.81; 95% confidence interval, 1.56-5.04; P < 0.001). Survivin(Hi)/B7-H1(+) tumors exhibited increased levels of infiltrating mononuclear cells and survivin-specific T cells compared with survivin(Low)/B7-H1(-) tumors. CONCLUSION: Patients with survivin(Hi)/B7-H1(+) ccRCC tumors are at increased risk of ccRCC death. Survivin(Hi)/B7-H1(+) tumors also harbor increased amounts of infiltrating mononuclear cells and survivin-specific T cells relative to survivin(Low)/B7-H1(-) tumors. Taken together, dual expression of survivin and B7-H1 can be used to predict ccRCC tumor aggressiveness.

B7-H4 expression in renal cell carcinoma and tumor vasculature: associations with cancer progression and survival.

B7-H4 is a recently described B7 family coregulatory ligand that has been implicated as an inhibitor of T cell-mediated immunity. Although expression of B7-H4 is typically limited to lymphoid cells, aberrant B7-H4 expression has also been reported in several human malignancies. To date, associations of B7-H4 with clinical outcomes for cancer patients are lacking. Therefore, we examined B7-H4 expression in fresh-frozen tumor specimens from 259 renal cell carcinoma (RCC) patients treated with nephrectomy between 2000 and 2003 and performed correlative outcome analyses. We report that 153 (59.1%) RCC tumor specimens exhibited B7-H4 staining and that tumor cell B7-H4 expression was associated with adverse clinical and pathologic features, including constitutional symptoms, tumor necrosis, and advanced tumor size, stage, and grade. Patients with tumors expressing B7-H4 were also three times more likely to die from RCC compared with patients lacking B7-H4 (risk ratio = 3.05; 95% confidence interval = 1.51-6.14; P = 0.002). Additionally, 211 (81.5%) specimens exhibited tumor vasculature endothelial B7-H4 expression, whereas only 6.5% of normal adjacent renal tissue vessels exhibited endothelial B7-H4 staining. Based on these findings, we conclude that B7-H4 has the potential to be a useful prognostic marker for patients with RCC. In addition, B7-H4 represents a target for attacking tumor cells as well as tumor neovasculature to facilitate immunotherapeutic treatment of RCC tumors. Last, we demonstrate that patients with RCC tumors expressing both B7-H4 and B7-H1 are at an even greater risk of death from RCC.

Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up.

B7-H1 participates in T-cell costimulation functioning as a negative regulator of immunity. Recent observations suggest that B7-H1 is expressed by renal cell carcinoma (RCC) tumor cells and is associated with poor prognosis. However, outcome analyses have been restricted to patients with fresh-frozen tissue and limited follow-up. We report the clinical effect of B7-H1 in RCC patients with a median of 10 years of follow-up. Between 1990 and 1994, 306 patients underwent nephrectomy for clear cell RCC and had paraffin tissue available for review. We did immunohistochemistry with anti-B7-H1 and conducted outcome analyses. Among the 306 patients, 73 (23.9%) harbored tumors with B7-H1 expression. Patients with tumor B7-H1 were at a significantly increased risk of both death from RCC [risk ratio (RR), 3.92; P < 0.001] and overall mortality (RR, 2.37; P < 0.001). The 5-year cancer-specific survival rates were 41.9% and 82.9% for patients with and without tumor B7-H1, respectively. In a multivariate model, tumor B7-H1 remained associated with cancer-specific death even after adjusting for tumor-node-metastasis stage, grade, and performance status (RR, 2.00; P = 0.003). In the subset of 268 patients with localized RCC, tumor B7-H1 was significantly associated with metastatic cancer progression (RR, 3.46; P < 0.001) and death from RCC (RR, 4.13; P < 0.001) even after adjusting for stage, grade, and performance status (RR, 1.78, P = 0.036). RCC patients with tumor B7-H1 are at significant risk of rapid cancer progression and accelerated rates of mortality. B7-H1 may function as a key determinant in RCC, abrogating immune responses directed against this immunogenic tumor.

B7-H1 glycoprotein blockade: a novel strategy to enhance immunotherapy in patients with renal cell carcinoma.

Cancer cell expression of the B7-H1 glycoprotein has been implicated as a potent inhibitor of T cell-mediated antitumoral immunity. We recently reported that B7-H1 is aberrantly expressed in renal cell carcinoma (RCC) and suggested that blockade of B7-H1, as demonstrated in several murine cancer models, represents a promising therapeutic target in RCC. Herein, we update our results with tumor-associated B7-H1 and discuss future clinical applications. Between 2000 and 2002, 196 patients underwent nephrectomy for clear-cell RCC and had fresh-frozen tissue available for review. Immunohistochemical analysis was performed on tumor cryosections, and outcome was obtained from the Mayo Clinic Nephrectomy Registry (Rochester, MN). At last follow-up 38 of the 196 patients died of RCC. The median duration of follow-up for patients still alive was 2.7 years. Among the 196 RCC specimens, 130 (66.3%) demonstrated aberrant tumor-associated B7-H1 expression. Patients with tumor-associated B7-H1 expression were significantly more likely to die of RCC compared with patients whose specimens did not have aberrant tumor-associated B7-H1 expression (risk ratio, 3.34; 95% confidence interval [CI], 1.30 - 8.55; P = 0.012). This risk persisted in multivariate analysis even after adjusting for the Mayo Clinic SSIGN (stage, size, grade, and necrosis) score (risk ratio, 3.52; 95% CI, 1.35-9.15; P = 0.010). Tumor-associated B7-H1 expression is significantly associated with poor prognosis among patients with clear-cell RCC. Manipulation of B7-H1 with therapeutic intent remains a realistic and viable therapeutic option.

Augmentation of T cell levels and responses induced by androgen deprivation.

Androgen has been implicated as a negative regulator of host immune function and a factor contributing to the gender dimorphism of autoimmunity. Conversely, androgen deprivation has been suggested to potentiate male host immunity. Studies have shown that removal of androgen in postpubertal male mice produces an increase in size and cellularity of primary and peripheral lymphoid organs, and enhances a variety of immune responses. Yet, few details are known about the effect of androgen removal on T cell-mediated immunity. In this study, we demonstrate two pronounced and independent alterations in T cell immunity that occur in response to androgen deprivation, provided by castration, in postpubertal male mice. First, we show that levels of T cells in peripheral lymphoid tissues of mice are increased by androgen deprivation. Second, T cells from these mice transiently proliferate more vigorously to TCR- and CD28-mediated costimulation as well as to Ag-specific activation. In addition, androgen deprivation accelerates normalization of host T and B cell levels following chemotherapy-induced lymphocyte depletion. Such alterations induced by androgen deprivation may have implications for enhancing immune responses to immunotherapy and for accelerating the recovery of the immune system following chemotherapy.

Silencing of secretin receptor function by dimerization with a misspliced variant secretin receptor in ductal pancreatic adenocarcinoma.

Secretin receptors that are key for regulation of healthy pancreatic ductal epithelial cells have been reported to be functionally absent on ductal pancreatic adenocarcinomas. Here, we examine the possible presence and function of molecular forms of the secretin receptor in pancreatic cancer cell lines and in primary tumors. Surprisingly, reverse transcription-PCR and sequencing demonstrated wild-type secretin receptor mRNA in each of four cell lines and three primary tumors. Lack of biological response to nanomolar concentrations of secretin was best explained by the demonstrated coexpression of a second and predominant transcript in each of the cell lines and tumors. This represented a variant of the secretin receptor in which the third exon was spliced out to eliminate residues 44-79 from the NH(2)-terminal tail. This spliceoform has only recently been recognized in a rare gastrinoma, where it was incapable of binding secretin or signaling, and possessed dominant-negative activity to suppress hormone action at the wild-type secretin receptor (1). Overexpression of wild-type secretin receptor in Panc-1 cells driven by transfection of fully processed cDNA resulted in normal responsiveness to low concentrations of secretin, establishing the ability of these cells to produce a receptor capable of normal biosynthesis, trafficking, and signaling. Bioluminescence resonance energy transfer demonstrated that the variant receptor could form a heterodimer with wild-type receptor, providing a molecular mechanism for its dominant-negative activity. This suggests that missplicing is responsible for expression of a secretin receptor variant having the ability to suppress the function of wild-type receptor by a direct interaction. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in receptor-bearing Chinese hamster ovary cells, the secretin receptor was shown to have growth-inhibitory effects. Suppression of this activity in pancreatic carcinoma might, therefore, facilitate tumor growth and progression of this aggressive neoplasm.

A misspliced form of the cholecystokinin-B/gastrin receptor in pancreatic carcinoma: role of reduced sellular U2AF35 and a suboptimal 3'-splicing site leading to retention of the fourth intron.

Abnormal splicing of primary RNA transcripts of normal genes is a recognized mechanism for the production of some abnormal proteins found in cancer cells. A misspliced form of the cholecystokinin-B/gastrin (CCK-B) receptor recently was reported to be present in colon carcinoma, where it was postulated to play a role in stimulating tumor growth (M. R. Hellmich et al., J. Biol. Chem., 275: 32122-32128, 2000). Here, we report the presence of the same abnormal protein in pancreatic carcinoma and explore the molecular basis for this missplicing event. Reverse transcription-PCR and sequencing were used to demonstrate the presence of a misspliced form of the CCK-B receptor having its fourth intron retained in three pancreatic cancer cell lines and in tumor tissue, but not in surrounding healthy pancreas, from two patients with pancreatic carcinoma. A mini-gene construct representing the region of this gene from its third through its fifth exon and containing the two intervening introns was produced and transiently expressed in the MIA PaCa-2 human pancreatic cancer cell line. Specific reverse transcription-PCR reactions with both vector-derived and receptor-specific primers demonstrated the presence of both correctly fully spliced and selectively misspliced forms of this receptor. Mutagenesis of the mini-gene demonstrated that a suboptimal sequence at the 3'-end of intron 4 contributed to this missplicing. This focused attention on the U2 small nuclear ribonucleoprotein particle auxiliary splicing factors (U2AFs) known to interact specifically with this domain. Indeed, quantitative real-time PCR demonstrated a reduced level of expression of one of these factors, U2AF35, in pancreatic cancer cells compared with healthy pancreas. Furthermore, the relative amount of missplicing of the CCK-B receptor mini-gene in the pancreatic cancer cell line was reversed by transfection of the cells with U2AF35 cDNA. This work describes the presence of an additional abnormal protein in pancreatic cancer and describes a new molecular mechanism for its production, providing additional potential therapeutic targets.