Antibiotic Treatment of Corynebacterium bovis-associated Clinical Disease in NSG Mice.

Depending on the strain of immunodeficient mice, Corynebacterium bovis infection can be asymptomatic or cause transient or prolonged skin disease. C. bovis infection of NOD. Cg- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice results in clinical skin disease that progresses in severity. Amoxicillin metaphylaxic and prophylaxic therapy prevents transmission and infection of mice after exposure to C. bovis and inhibits the growth of C. bovis isolates at therapeutic doses that are clinically achievable in mice. Amoxicillin is not efficacious for treatment of transient clinical skin disease in athymic nude mice, but the efficacy of amoxicillin treatment has not previously been characterized in C. bovis -infected NSG mice. In the current study, NSG mice were treated with amoxicillin beginning at 5 wk after exposure to C. bovis, at which time they had well-established clinical signs of disease. Clinical signs were scored to assess disease progression, regression, and reappearance. Our results showed that amoxicillin treatment for 3 or 6 wk reduced the clinical scores of NSG mice with C. bovis -associated clinical disease. In addition, withdrawal of treatment led to the recurrence of clinical signs. Collectively, our data suggest that amoxicillin treatment is effective in alleviating the clinical signs associated with C. bovis infection for the duration of treatment in NSG mice. Clinical intervention with antibiotics for C. bovis -infected NSG mice can be an option for management of C. bovis -related clinical disease either before or during facility-wide remediation efforts.

Fibronectin Contributes to a BRAF Inhibitor-driven Invasive Phenotype in Thyroid Cancer through EGR1, Which Can Be Blocked by Inhibition of ERK1/2.

Mutations in BRAF are common in advanced papillary and anaplastic thyroid cancer (PTC and ATC). However, patients with BRAF-mutant PTC currently lack therapies targeting this pathway. Despite the approved combination of BRAF and MEK1/2 inhibition for patients with BRAF-mutant ATC, these patients often progress. Thus, we screened a panel of BRAF-mutant thyroid cancer cell lines to identify new therapeutic strategies. We showed that thyroid cancer cells resistant to BRAF inhibition (BRAFi) exhibit an increase in invasion and a proinvasive secretome in response to BRAFi. Using reverse-phase protein array (RPPA), we identified a nearly 2-fold increase in expression of the extracellular matrix protein, fibronectin, in response to BRAFi treatment, and a corresponding 1.8- to 3.0-fold increase in fibronectin secretion. Accordingly, the addition of exogenous fibronectin phenocopied the BRAFi-induced increase in invasion while depletion of fibronectin in resistant cells resulted in loss of increased invasion. We further showed that BRAFi-induced invasion can be blocked by inhibition of ERK1/2. In a BRAFi-resistant patient-derived xenograft model, we found that dual inhibition of BRAF and ERK1/2 slowed tumor growth and decreased circulating fibronectin. Using RNA sequencing, we identified EGR1 as a top downregulated gene in response to combined BRAF/ERK1/2 inhibition, and we further showed that EGR1 is necessary for a BRAFi-induced increase in invasion and for induction of fibronectin in response to BRAFi. IMPLICATIONS: Together, these data show that increased invasion represents a new mechanism of resistance to BRAF inhibition in thyroid cancer that can be targeted with an ERK1/2 inhibitor.

A Clinical Scoring Systems for the Evaluation of Corynebacterium bovis -associated Disease in NSG Mice.

Clinical signs of Corynebacterium bovis infections are well-known in athymic nude mice. However, C. bovis can also infect and cause clinical signs in many hirsute, immunocompromised mouse strains such as NSG (NOD. Cg-Prkdcscid Il2rgtm1Wgl/SzJ). Typically, the clinical assessment of C. bovis-infected mice begins when overt clinical signs are initially observed and thus the early course of infection has not been thoroughly described. The goal of this study was to characterize the clinical progression of C. bovis infection in NSG mice under experimental conditions and develop a quantifiable clinical scoring system. For the development and application of this clinical scoring system, 54 naïve NSG mice were exposed to soiled bedding from clinically ill C. bovis-infected NSG mice and the emergence of clinical signs was monitored and scored weekly for 8 wk. Overall, we identified 6 benchmark changes associated with C. bovis clinical infection. Four changes were the appearance of the eyes, ears, hair coat, and posture. Two behavioral changes were increased grooming activity and rapid head shaking. All clinical signs appeared consistently and progressed temporally with increasing clinical severity. Characterization of clinical signs and scoring of clinical disease will aid veterinarians in the assessment of C. bovis-infected NSG mice and may help in the evaluation of current and future clinical interventions used to prevent or treat C. bovis-infected immunodeficient mice.

Metaphylactic Antibiotic Treatment to Prevent the Transmission of Corynebacterium bovis to Immunocompromised Mouse Offspring.

Current methods for eradicating Corynebacterium bovis, such as depopulation, embryo transfer, and cesarean rederivation followed by cross fostering, are expensive, complex, and time-consuming. We investigated a novel method to produce immunocompromised offspring free of C. bovis from infected NOD. Cg-PrkdcscidIl2rgtm1Wgl/SzJ (NSG) breeding pairs. Adult NSG mice were infected with C. bovis, paired, and randomly assigned to either a no-antibiotic control group (NAB, n = 8) or a group that received amoxicillin-clavulanic acid (0.375 mg/mL) in their drinking water for a mean duration of 7 wk (AB group, n = 7), spanning the time from pairing of breeders to weaning of litters. The AB group also underwent weekly cage changes for 3 wk after pairing to decrease intracage C. bovis contamination, whereas the NAB mice received bi-weekly cage changes. Antibiotics were withdrawn at the time of weaning. All litters (n = 7) in the AB group were culture- and qPCR-negative for C. bovis and remained negative for the duration of the study, whereas all litters in the NAB group (n = 6) remained C. bovis positive. A single adult from each breeding pair was sampled at weaning and at 5 and 10 wk after weaning to confirm the maintenance of (NAB) or to diagnose the reemergence (AB) of C. bovis infection. By the end of the study, C. bovis infection had returned in 3 of the 7 (43%) tested AB adults. Our data suggest that metaphylactic antibiotic use can decrease viable C. bovis organisms from adult breeder mice and protect offspring from infection. However, using antibiotics with frequent cage changing negatively affected breeding performance. Nevertheless, this technique can be used to produce C. bovis-free NSG offspring from infected adults and may be an option for salvaging infected immunocompromised strains of mice that are not easily replaced.

Diagnosis, Surveillance and Management of Streptococcus equi subspecies zooepidemicus Infections in Chinchillas (Chinchilla lanigera).

During a 6-mo period, two 5-6 mo old female chinchillas (Chinchilla lanigera) were examined at the University of Colorado Anschutz Medical Campus after the discovery of firm, nonmobile masses in the left ventral cervical and left axillary region. Other than these findings and mild weight loss, both chinchillas' physical exams were normal. Bloodwork revealed an inflammatory leukogram characterized by leukocytosis, toxic neutrophils, lymphopenia, and monocytosis with mild, nonregenerative anemia. At necropsy, both masses were identified as abscesses. Streptococcus equi, subspecies zooepidemicus (S. zooepidemicus) was isolated in pure culture. Histology of the lungs, liver, spleen, and kidneys showed a marked increase in the numbers of both polymorphonuclear leukocytes and lymphocytes. Both animals were deemed unsuitable for research and were euthanized under isoflurane anesthesia by an intracardiac injection of pentobarbital sodium solution. S. zooepidemicus is an opportunistic, commensal organism found in the upper respiratory tract of horses. This organism has been documented to cause disease in other species and is zoonotic. Infections in humans have been reported, resulting in glomerulonephritis, endocarditis, septic arthritis, osteomyelitis, meningitis, and death. To aid in diagnosis and prospective surveillance of this bacteria, oral and nasal swabs were collected from the remaining cohort of chinchillas, and a qPCR screening assay was implemented. Within 12 mo, 4 of 41 additional females tested positive by culture or qPCR, resulting in a disease prevalence of 14% (6 of 43). However, only 2 of the additional 4 S. zooepidemicus positive animals developed clinical signs. The potential for the spread of infection, zoonosis, and adverse effects on research demonstrate that surveillance for S. zooepidemicus should be considered in a biomedical research environment.

Establishment and Characterization of Four Novel Thyroid Cancer Cell Lines and PDX Models Expressing the RET/PTC1 Rearrangement, BRAFV600E, or RASQ61R as Drivers.

Cancer cell lines are critical models to study tumor progression and response to therapy. In 2008, we showed that approximately 50% of thyroid cancer cell lines were redundant or not of thyroid cancer origin. We therefore generated new authenticated thyroid cancer cell lines and patient-derived xenograft (PDX) models using in vitro and feeder cell approaches, and characterized these models in vitro and in vivo. We developed four thyroid cancer cell lines, two derived from 2 different patients with papillary thyroid cancer (PTC) pleural effusions, CUTC5, and CUTC48; one derived from a patient with anaplastic thyroid cancer (ATC), CUTC60; and one derived from a patient with follicular thyroid cancer (FTC), CUTC61. One PDX model (CUTC60-PDX) was also developed. Short tandem repeat (STR) genotyping showed that each cell line and PDX is unique and match the original patient tissue. The CUTC5 and CUTC60 cells harbor the BRAF (V600E) mutation, the CUTC48 cell line expresses the RET/PTC1 rearrangement, and the CUTC61 cells have the HRAS (Q61R) mutation. Moderate to high levels of PAX8 and variable levels of NKX2-1 were detected in each cell line and PDX. The CUTC5 and CUTC60 cell lines form tumors in orthotopic and flank xenograft mouse models. IMPLICATIONS: We have developed the second RET/PTC1-expressing PTC-derived cell line in existence, which is a major advance in studying RET signaling. We have further linked all cell lines to the originating patients, providing a set of novel, authenticated thyroid cancer cell lines and PDX models to study advanced thyroid cancer.

Resistance to Src inhibition alters the BRAF-mutant tumor secretome to promote an invasive phenotype and therapeutic escape through a FAK>p130Cas>c-Jun signaling axis.

Few therapy options exist for patients with advanced papillary and anaplastic thyroid cancer. We and others have previously identified c-Src as a key mediator of thyroid cancer pro-tumorigenic processes and a promising therapeutic target for thyroid cancer. To increase the efficacy of targeting Src in the clinic, we sought to define mechanisms of resistance to the Src inhibitor, dasatinib, to identify key pathways to target in combination. Using a panel of thyroid cancer cell lines expressing clinically relevant mutations in BRAF or RAS, which were previously developed to be resistant to dasatinib, we identified a switch to a more invasive phenotype in the BRAF-mutant cells as a potential therapy escape mechanism. This phenotype switch is driven by FAK kinase activity, and signaling through the p130Cas>c-Jun signaling axis. We have further shown this more invasive phenotype is accompanied by alterations in the secretome through the increased expression of pro-inflammatory cytokines, including IL-1ß, and the pro-invasive metalloprotease, MMP-9. Furthermore, IL-1ß signals via a feedforward autocrine loop to promote invasion through a FAK>p130Cas>c-Jun>MMP-9 signaling axis. We further demonstrate that upfront combined inhibition of FAK and Src synergistically inhibits growth and invasion, and induces apoptosis in a panel of BRAF- and RAS-mutant thyroid cancer cell lines. Together our data demonstrate that acquired resistance to single-agent Src inhibition promotes a more invasive phenotype through an IL-1ß>FAK>p130Cas>c-Jun >MMP signaling axis, and that combined inhibition of FAK and Src has the potential to block this inhibitor-induced phenotype switch.

Procedure for Horizontal Transfer of Patient-Derived Xenograft Tumors to Eliminate Corynebacterium bovis.

Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as a model for cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retain many of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strains used to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At our institution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infected with C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfully harvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our data demonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfully be used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.

Detection and Elimination of Corynebacterium bovis from Barrier Rooms by Using an Environmental Sampling Surveillance Program.

Rodent health-monitoring programs based on sampling an IVC system's exhaust air dust (EAD) has enhanced and even replaced traditional sentinels for some rodent pathogens. EAD testing by qPCR assay is an optimal surveillance method for the rapid detection of Corynebacterium bovis-infected immunodeficient mice. Here we demonstrate that an active EAD surveillance program for C. bovis can be used to maintain nude mice C. bovis-free after the transition from historically enzootically infected colonies. During 3 events over 3 y, rapid detection of infection, elimination of infected mice, aggressive quarantine measures, and local decontamination prevented the spread of C. bovis within 2 barrier rooms. In total, 4 cages of infected nude mice were identified and removed, preventing the spread of infection to 469 other cages of immunodeficient mice. In addition, we present data regarding a refinement to EAD testing which enables row-specific surveillance of an IVC rack. This technique systemically decreases the amount of testing required to locate an individually infected cage. Due to our ability to rapidly detect and localize an infected cage, we were able to investigate the route of C. bovis introduction into our barrier rooms. Our epidemiologic investigation suggested that the transmission of C. bovis occurred through contaminated, cryopreserved, patient-derived xenograft tumor tissue. This previously unknown source of C. bovis can infect mice used to propagate these tumors. Together, these data demonstrate that a remediation program that combines rapid detection, test-and-cull, and local decontamination under quarantine conditions can eliminate C. bovis from a mouse colony.

Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds.

Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks' air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection.

A rexinoid antagonist increases the hypothalamic-pituitary-thyroid set point in mice and thyrotrope cells.

Retinoid X receptor (RXR) signaling influences thyrotrope function. Synthetic RXR agonists, rexinoids, can cause central hypothyroidism. To test the hypothesis that endogenous rexinoids contribute to the TSH 'set point', TαT1 mouse thyrotrope cells were treated with a rexinoid antagonist, LG101208. Increasing concentrations of LG101208 significantly increased TSHß mRNA levels, indicating that the rexinoid antagonist may interfere with RXR-signaling by an endogenous rexinoid in thyrotropes. When the same experiments were repeated in the presence of charcoal-stripped serum the effect of the rexinoid antagonist was lost. Pretreatment with the transcription inhibitor DRB blocked the increase of TSHß mRNA levels by rexinoid antagonist, indicating the primary effect is at the level of gene transcription. Mice treated with LG101208 had higher levels of serum T4, T4/TSH ratios as well as pituitary α-subunit and TSHß mRNA compared with vehicle treated mice. Hypothalamic TRH levels were unchanged. In summary, the rexinoid antagonist, LG101208, increases TSH subunit mRNA levels in thyrotrope cells and mouse pituitaries, primarily at the level of gene transcription. These data suggest that an "endogenous rexinoid" contributes to the TSH 'set point' in thyrotropes.

Retinoid and thiazolidinedione therapies in melanoma: an analysis of differential response based on nuclear hormone receptor expression.

BACKGROUND: Metastatic melanoma has a high mortality rate and suboptimal therapeutic options. Molecular targeting may be beneficial using the rexinoid LGD1069, a retinoid x receptor selective agonist, and thiazolidinediones (TZD), PPARgamma selective ligands, as novel treatments. RESULTS: Mouse xenograft models with human melanoma cell lines [A375(DRO) or M14(5-16)] were treated for 4 weeks with daily vehicle, RXR agonist (rexinoid, LGD1069, 30 mg/kg/d), PPARgamma agonist (TZD, rosiglitazone, 10 mg/kg/d) or combination. A375(DRO) tumor growth was significantly inhibited by either ligand alone and the combination had an additive effect. M14(5-16) tumors only responded to LGD1069 100 mg/kg/day. A375(DRO) sublines resistant to rexinoid, TZD and combination were generated and all three sublines had reduced PPARgamma expression but preserved RXR expression. shRNA knockdown of PPARgamma or RXRgamma attenuated the rexinoid, TZD and combination ligand-mediated decreased proliferation in A375(DRO) cells. Rexinoid (LGD1069) and retinoid (TTNPB) treatment of M14(5-16) cells resulted in decreased proliferation that was additive with combination of both rexinoid and retinoid. shRNA knockdown of RXRgamma resulted in a decreased response to either ligand. CONCLUSION: A375 (DRO) melanoma cell growth is inhibited by rexinoid and TZD treatment, and this response is dependent on RXR and PPARgamma receptor expression. M14 (5-16) melanoma cell growth is inhibited by rexinoid and retinoid treatment, and this response is dependent on RXR expression. These findings may help guide molecular-based treatment strategies in melanoma and provide insight for mechanisms of resistance to nuclear receptor targeted therapies in certain cancers.

Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification.

CONTEXT: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. OBJECTIVE: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. RESULTS: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. CONCLUSIONS: Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

In vivo and microarray analysis of rexinoid-responsive anaplastic thyroid carcinoma.

PURPOSE: Anaplastic thyroid carcinoma is rare, yet lethal despite aggressive therapy. Molecular targeting may be beneficial using the rexinoid LGD1069, a retinoid X receptor-selective agonist, as a novel treatment. In this report, we describe the efficacy of LGD1069 in anaplastic thyroid carcinoma in vitro and assess the in vivo treatment effects on a responsive cancer. Additionally, we explore potential mediators of the rexinoid effect on a responsive anaplastic thyroid cancer using comparative microarray analysis. EXPERIMENTAL DESIGN: Anaplastic thyroid cancer cell lines DRO, ARO, and FRO were treated with LGD1069 in vitro. Responsive DRO xenograft tumors were treated with control chow or chow containing a low dose (30 mg/kg/d) or a high dose (100 mg/kg/d) of LGD1069. Comparative microarray analysis of DRO cells treated with LGD1069 compared with volume-equivalent control was assessed after 24 h of treatment to evaluate early gene expression changes. RESULTS: DRO xenograft tumor growth was inhibited by LGD1069 treatment in a dose-dependent manner. Comparative microarray analysis showed that 80 genes had a significant increase in expression and 29 genes had a decrease in expression after 24 h of treatment with LGD1069. Expression of angiopoietin-like 4 (ANGPTL4) mRNA was increased 6.5-fold. A trend towards an increase in ANGPTL4 mRNA (not statistically significant) was seen in treated tumors in vivo and this correlated with decreased tumor vascularity and increased necrosis. CONCLUSIONS: LGD1069 therapy decreases proliferation in an anaplastic thyroid cancer cell line that expresses retinoid X receptor-gamma, and this effect is confirmed with decreased tumor size in vivo in a nude mouse model. ANGPTL4 is increased in DRO in response to LGD1069 and may be a potential mediator of the effects of rexinoid treatment.

Effects of rexinoids on thyrotrope function and the hypothalamic-pituitary-thyroid axis.

Retinoid X receptor (RXR)-selective retinoids (rexinoids) can cause central hypothyroidism in humans, and this effect has been confirmed in rodent models. In this report, we characterized the effect of rexinoids on the hypothalamic-pituitary-thyroid axis in mice and TSH regulation in a thyrotrope-derived cell line. The synthetic rexinoid (LG 268) suppressed TSH and T4 levels in mice. Hypothalamic TRH mRNA was unaffected, but steady-state pituitary TSHbeta mRNA levels were significantly lowered, suggesting a direct effect of rexinoids on thyrotropes. LG 268 suppressed TSH protein secretion and TSHbeta mRNA in TalphaT1 thyrotropes as early as 8 h after treatment, whereas the retinoic acid receptor-selective retinoid (TTNPB) had no effect. Type 2 iodothyronine deiodinase (D2) mRNA and activity were suppressed by LG 268 in TalphaT1 cells, whereas only D2 mRNA was suppressed in mouse pituitaries. LG 268 suppressed TSHbeta promoter activity by 42% and the -200 to -149 region accounted for a majority of the LG 268-mediated suppression of promoter activity. The RXRgamma isotype is expressed in thyrotropes. In vitro transfection and in vivo transgenic studies indicate that any RXR isotype can mediate TSH suppression by rexinoids, but the RXRgamma isotype is most efficient at mediating this response. RXRgamma-deficient mice lacked pituitary D2 mRNA suppression by LG 268, but D2 activity remained intact. In summary, RXR-selective retinoids (rexinoids) have multiple effects on the hypothalamic-pituitary-thyroid axis. Rexinoids directly suppress TSH secretion, TSHbeta mRNA levels and promoter activity, and D2 mRNA levels but have no direct effect on hypothalamic TRH levels. Rexinoids also stimulate type 1 iodothyronine deiodinase activity in the liver and pituitary.

Retinoic acid and retinoid X receptors are differentially expressed in thyroid cancer and thyroid carcinoma cell lines and predict response to treatment with retinoids.

Therapy for patients with advanced thyroid carcinoma is limited. Clinical and in vitro studies suggest that some patients with advanced thyroid cancer may respond to therapy with retinoic acid. mRNA expression of the six retinoic acid (RAR) and retinoid X receptor (RXR) isoforms (RARalpha, -beta, -gamma and RXRalpha, -beta, -gamma) was measured in four human thyroid cell lines, and protein expression was subsequently measured in 10 thyroid cancer cell lines. Two isoforms, RARbeta and RXRgamma, were differentially expressed in the four cell lines. Comparison of 10 thyroid tumors and matched normal thyroid tissue confirmed differential tumor expression of RARbeta and RXRgamma and lack of the RXRgamma isoform in normal thyroid tissue. Cell lines expressing both RARbeta and RXRgamma demonstrated significant growth suppression when treated with retinoids, whereas cell lines lacking these isoforms were unaffected. Expression of RARbeta, the isoform associated with suppression of tumor growth in other cancer types, was not affected by treatment with retinoids in the thyroid cancer cell lines. LG346 increased apoptosis and decreased cells in the S-phase in an anaplastic carcinoma cell line, suggesting that this retinoid causes growth suppression of these cells by multiple mechanisms. In summary, we identified the RARbeta and RXRgamma isoform to be differentially expressed in thyroid cancer cell lines and tumor tissue. These isoforms seem to predict response to retinoid therapy in thyroid cancer cell lines.

Correlation of survivin mRNA detection with histologic diagnosis in normal endometrium and endometrial carcinoma.

BACKGROUND: The objective of this study was to determine if survivin mRNA expression is a marker of endometrioid adenocarcinoma and if survivin mRNA levels correlate with tumor grade and stage. METHODS: Twenty-six samples of endometrioid adenocarcinoma and 18 cases of benign endometrium were obtained at surgery. RNA was extracted from tissues and was used for quantitative real-time RT-PCR, targeting a 91-bp sequence of survivin mRNA and the levels were standardized to the levels of ribosomal RNA. Statistical analysis of the correlation between histologic diagnosis and the corrected survivin mRNA levels was performed by the Fisher Exact test and the Kruskal-Wallis test. RESULTS: Survivin mRNA was detected in all specimens. Survivin mRNA levels were increased in proliferative endometrium (P = 0.0509) and was increased in correlation with ascending grade in endometrioid adenocarcinoma (P = 0.01). CONCLUSION: Survivin mRNA is not a specific marker of endometrial cancer, but may reflect an important mechanism in tumor progression of the endometrial mucosa.

Quantitative analysis of telomerase hTERT mRNA and telomerase activity in endometrioid adenocarcinoma and in normal endometrium.

OBJECTIVES: In the current study, the quantitative levels of telomerase hTERT mRNA and the functional telomerase repeat amplification protocol (TRAP) assay were correlated with tumor grade in endometrial carcinomas and with the histologic phase of normal endometrium. METHODS: Twenty-six samples of endometroid adenocarcinoma and 20 cases of benign endometrium were obtained from hysterectomy specimens. Total RNA was extracted from each tissue sample and used for quantitative real-time RT-PCR of hTERT mRNA and the levels were standardized to the levels of ribosomal RNA. Quantitative determination of telomerase activity was performed by the polymerase chain-based TRAP assay and the levels of expression were defined by the ratio of radioactivity incorporated into the 6-bp telomerase amplification products versus the radioactivity incorporated into an internal standard (telomerase/ITAS x 100 = 1 RU). Statistical analyses were performed using the Fisher exact test or chi2 test, a Wilcoxon rank sum test, and a linear regression analysis. RESULTS: hTERT mRNA and telomerase activity levels showed a linear association in the study group (P = 0.006, R2 = 0.139). hTERT mRNA levels and telomerase activity levels were significantly higher in endometrial cancer (179 pg/ng rRNA, 44 relative units (RU)) than in normal endometrium (45 pg/ng), (15 RU) (P = 0.009, P = 0.006). In normal endometrium, hTERT mRNA and telomerase activity levels were highest in the proliferative phase (74 pg/ng rRNA, 25 RU) and were relatively low in secretory (13 pg/ng rRNA, 6 RU) and atrophic endometrium (9 pg/ng rRNA, 2 RU). CONCLUSION: These results suggest that the quantitative analysis of hTERT and telomerase activity may have potential roles as diagnostic or prognostic adjuncts for both premenopausal and postmenopausal patients with endometrial cancer.