bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial functional impairment with aging is exaggerated in isolated mitochondria compared to permeabilized myofibers
Authors: Picard Martin , Ritchie Darmyn , Wright Kathryn J. , Romestaing Caroline , Thomas Melissa M. , Rowan Sharon L. , Taivassalo Tanja , Hepple Russell T.
Published: September 2010

Mitochondria regulate cellular bioenergetics and apoptosis and have been implicated in aging. However, it remains unclear whether age‐related loss of muscle mass, known as sarcopenia, is associated with abnormal mitochondrial function. Two technically different approaches have mainly been used to measure mitochondrial function: isolated mitochondria and permeabilized myofiber bundles, but the reliability of these measures in the context of sarcopenia has not been systematically assessed before. A key difference between these approaches is that contrary to isolated mitochondria, permeabilized bundles contain the totality of fiber mitochondria where normal mitochondrial morphology and intracellular interactions are preserved. Using the gastrocnemius muscle from young adult and senescent rats, we show marked effects of aging on three primary indices of mitochondrial function (respiration, H2O2 emission, sensitivity of permeability transition pore to Ca2+) when measured in isolated mitochondria, but to a much lesser degree when measured in permeabilized bundles. Our results clearly demonstrate that mitochondrial isolation procedures typically employed to study aged muscles expose functional impairments not seen in situ. We conclude that aging is associated with more modest changes in mitochondrial function in sarcopenic muscle than suggested previously from isolated organelle studies.
Predictors of Nursing Home Residents' Time to Hospitalization
Authors: O'Malley A. James , Caudry Daryl J. , Grabowski David C.
Published: September 2010

Objectives. To model the predictors of the time to first acute hospitalization for nursing home residents, and accounting for previous hospitalizations, model the predictors of time between subsequent hospitalizations.

Data Sources. Merged file from New York State for the period 1998–2004 consisting of nursing home information from the minimum dataset and hospitalization information from the Statewide Planning and Research Cooperative System.

Study Design. Accelerated failure time models were used to estimate the model parameters and predict survival times. The models were fit to observations from 50 percent of the nursing homes and validated on the remaining observations.

Principal Findings. Pressure ulcers and facility‐level deficiencies were associated with a decreased time to first hospitalization, while the presence of advance directives and facility staffing was associated with an increased time. These predictors of the time to first hospitalization model had effects of similar magnitude in predicting the time between subsequent hospitalizations.

Conclusions. This study provides novel evidence suggesting modifiable patient and nursing home characteristics are associated with the time to first hospitalization and time to subsequent hospitalizations for nursing home residents.
Curative‐intent radiation therapy as a treatment modality for appendicular and axial osteosarcoma: a preliminary retrospective evaluation of 14 dogs with the disease
Authors: Walter C. U. , Dernell W. S. , LaRue S. M. , Lana S. E. , Lafferty M. H. , LaDue T. A. , Withrow S. J.
Published: September 2010

Canine osteosarcoma is a common bone malignancy associated with aggressive local disease and rapid metastasis. Current local therapeutic modalities do not provide curative‐intent options for dogs with significant orthopaedic or neurologic disease, dogs which are denied amputation or dogs with non‐resectable lesions. The goals of this retrospective study included the evaluation of local control, survival, and time to the development of metastases in 14 dogs treated with curative‐intent radiation therapy and chemotherapy. Median local disease control was 202 days (79–777). Median survival was 209 days (79–781). Median time to metastasis was 314 days (7–645). No significant correlation was found between the outcome and pre‐treatment alkaline phosphatase levels, radiographic appearance, tumour site, radiation dose or chemotherapeutics administered. In these dogs, full‐course radiation therapy in conjunction with chemotherapy was not found to yield equivalent results to the standard of care options.
Comparative Oncology Solution to Understanding Hormonal Impact in the Epidemiology of Lymphoma
Authors: Henry C. J. , Hahn A. W. , Tyler J. W. , Steinberg H. S. , Caldwell C. W.
Published: September 2010

Introduction:  Surveillance, Epidemiology & End Results (SEER) data indicate that non‐Hodgkin's lymphoma (NHL) is approximately 50% more common among men than women. A similar male predisposition is reported for canine lymphoma, yet the underlying role of gender in lymphoma etiology remains elusive. Because similarities exist between canine lymphoma and NHL, databases including the Veterinary Medical Database (VMDB) and Veterinary Cancer Registry (VCR) may prove useful in understanding lymphoma epidemiology. This study sought to determine the relationship between gender and development of canine lymphoma.

Materials:  Data from 1980 to 2000 were retrieved from the VMDB and sorted by gender and reproductive status. Spayed or neutered dogs diagnosed with lymphoma were compared to intact dogs seen each year in each gender category using a two‐tailed Student's t‐test. The VCR was searched for all canine lymphoma cases. The number of cases in each gender group was compared to the number of cancer diagnoses per group using a Chi‐square test (3 degrees of freedom). Differences were deemed significant when P < 0.050.

Results:  The VMDB included 15,091 lymphoma cases in a population of 1.34 million dogs. In the VCR database, 394 lymphoma cases were identified amongst 6,070 canine cancer diagnoses. In both analyses, intact females were significantly less likely to develop lymphoma than were other gender groups.

Conclusions:  In dogs, as in people, female gender and intact reproductive status appears to be protective against the development of lymphoma. This data suggests that further examination of the role of estrogen in lymphoma prevention is warranted.
Targeting the RNA component of Telomerase Through RNA Interference and the Development of Vector Delivery Systems
Authors: Paoloni M. , Gunderman J. R. , Estelle L. N. , Padilla M. , Argyle D. J.
Published: September 2010

From studies on cellular senescence and cancer, Telomerase has emerged as a near universal marker of malignancy which gives rise to the immortal phenotype of cancer. The Telomerase enzyme is composed minimally of an RNA template component and a, catalytic component, reverse transcriptase. We have previously demonstrated that RNA interference can be used to block the reverse transcriptase component at the mRNA level. In this study we used the same RNAi technology to target the RNA component, with the overall goal of improving the efficiency of cell killing.

Three simple interfering RNA molecules (siRNA) targeted to the canine telomerase RNA component were generated. Simple transfection studies in telomerase positive canine cells (HSA SB) demonstrated rapid telomerase inhibition and cell killing when compared to control cells. Using the template design of one of the siRNA molecules, two vector systems (adenovirus and lentivirus) were constructed. Cell transduction experiments demonstrated these viral technologies as efficient delivery vehicles. These studies demonstrate the efficiency of RNAi technologies and offer the development of vector systems for in vivo cancer therapy.
Immunohistochemical Differentiation and Clinicopathological Features of Canine Gastrointestinal Stromal Tumors and Leiomyosarcomas
Authors: Russell K. N. , Mehler S. J. , Skorupski K. A. , Baez J. L. , Shofer F. S. , Goldschmidt M. H.
Published: September 2010

Introduction:  Gastrointestinal stromal tumors (GIST) and leiomyosarcomas (LMS) have recently been differentiated by immunohistochemical staining techniques and have been shown to have different biological behaviors in humans. Expression of the c‐kit protein, a transmembrane tyrosine kinase receptor, occurs in nearly all GISTs. The aim of this study was to differentiate canine GIST from LMS, and to compare their clinicopathological features.

Methods:  Archived blocks of previously diagnosed LMS were analyzed. Immunohistochemical staining using antibodies against c‐kit, smooth muscle actin (SMA), desmin, vimentin and S100 was performed. GISTs were diagnosed based on positive c‐kit staining. LMS were diagnosed based on absence of c‐kit staining and positive SMA staining. Follow‐up information was obtained from medical records and telephone interviews with owners.

Results:  Forty‐two dogs were included in the study. Mean age was 10.9 yrs (range 5–15 yrs). There were 18 females and 24 males. Twenty‐eight tumors were GISTs, 10 were LMS and 4 stained negatively for c‐kit, SMA and S100 (sarcomas). GISTs were more likely to occur in the large intestine and LMS were more common in the small intestine (p = 0.01). All were surgically excised and only two were treated with adjunctive chemotherapy. Only two GISTs and one sarcoma had metastasized at the time of surgery. Survival time in dogs discharged after surgery for GIST, LMS and sarcomas was 1123, 233 and 88 days respectively (p = 0.08).

Conclusions:  Many previously diagnosed LMS should be reclassified as GIST based on the results of immunohistochemical staining. The biological behavior of these tumors appears to be different.
Effects of Mycobacterial Cell Wall‐DNA Complex (MCC), Alendronate and Pamidronate on Canine Osteosarcoma Cell Lines
Authors: Filion M. C. , Filion B. , Phillips N. C.
Published: September 2010

Introduction:  MCC, a cell wall composition prepared from Mycobacterium phlei., inhibits the proliferation and induces apoptosis in a wide range of tumor cells. Bisphosphonates have been reported to inhibit the proliferation of canine osteosarcoma cell lines. In this study, we have determined the activity of MCC alone and in combination with the bisphosphonates alendronate and pamidronate on canine osteosarcoma cell lines.

Methods:  Canine osteosarcoma cell lines D17 and D22 were incubated with different concentrations of MCC (0.01–100 μg/ml) and suboptimal concentrations of alendronate and pamidronate for 72 hours. Cellular proliferation was measured by MTT reduction. Nuclear DNA condensation was determined using with Hoescht 33258 staining, and apoptosis by flow cytometry using active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies.

Results:  MCC inhibited the proliferation of both canine osteosarcoma D17 and D22 cell lines in a concentration‐dependent manner. The IC50 for D17 cells was 3.9 μg/ml and for D22 cells 44.4 μg/ml. Cells incubated with 100 μg/ml MCC were positive for Hoescht staining, active caspase‐3 and cleaved PARP, indicative of cell death by apoptosis. The addition of alendronate or pamidronate was found to potentiate the apoptosis‐inducing activity of MCC. Maximal activity was observed when 5 μM alendronante or 10 μM pamidronate were used in combination with 100 μg/ml MCC.

Conclusion:  MCC inhibits the proliferation and induces apoptosis in canine osteosarcoma cell lines in vitro. This anticancer activity can be potentiated by the use of alendronate and pamidronate. These data support the development of MCC as a therapeutic agent for the treatment of canine osteosarcoma.
Phase I & Ib Trials of Murine Tyrosinase ± Human GM‐CSF DNA Vaccination in Dogs with Advanced Malignant Melanoma
Authors: Bergman P. J. , Camps‐Palau M. A. , McKnight J. A. , Leibman N. F. , Novosad C. A. , Brenn S. , Ettinger S. N. , Endicott M. M. , Finora K. , Craft D. , Leung C. , Liao J. , Riviere I. , Hohenhaus A. E. , Houghton A. , Perales M. , Wolchok J. D.
Published: September 2010

Introduction:  Canine malignant melanoma (CMM) is an aggressive neoplasm treated with surgery and/or fractionated RT; however, metastatic disease is common and chemoresistant. Preclinical and clinical studies by our laboratory and others have shown that xenogeneic DNA vaccination with tyrosinase family members can produce immune responses resulting in tumor rejection or protection and prolongation of survival. The potency of DNA vaccines can be further enhanced by adding DNA encoding cytokine genes. We have shown in preclinical mouse models that GM‐CSF DNA enhances immune responses and tumor protection. These studies provided the impetus for murine tyrosinase (muTyr) ± human GM‐CSF (huGM‐CSF) DNA vaccination in CMM.

Materials & Methods:  Two groups of five dogs each with advanced (WHO stage II‐IV) CMM received four biweekly IM injections (100 ug or 500 ug, respectively/vaccination) of plasmid DNA encoding muTyr via the Biojector2000 jet delivery device. Subsequently, three groups of nine dogs each with advanced CMM received four biweekly IM injections of plasmid DNA encoding muTyr (50 ug), huGM‐CSF (3 dogs each at 100/400/800 ug) or both.

Results:  Minimal to mild pain was noted on vaccination and no toxicity or induction of autoimmunity was seen. The KM median survival time was 224 days (100/500 ug muTyr), 278 days (50 ug muTyr), 140 days (huGM‐CSF) and >265 days (muTyr & huGM‐CSF; 6 dogs still alive).

Conclusions:  The results of these trials demonstrate that xenogeneic DNA vaccination continues to be a safe and potentially therapeutic modality for CMM. These results also warrant further evaluation of this novel therapeutic in a Phase II setting.
Retropective Evaluation of Survival Time of Dogs with Stage III Osteosarcoma that Undergo Treatment
Authors: Boston S. , Ehrhart N. , Dernell W. , Lafferty M. , Withrow S.
Published: September 2010

Introduction:  Stage III osteosarcoma generally carries a grave prognosis. Despite this, some owners elect to treat using palliative or curative intent protocols. The purpose of this study was to determine the survival times for dogs with stage III osteosarcoma that undergo treatment and to evaluate the variables that affect survival time.

Methods:  Retrospective study using the CSU Animal Cancer Center osteosarcoma data base. Search criteria included dogs diagnosed with osteosarcoma 1985–2004 with metastasis at the time of presentation. Dogs were excluded if they were euthanized at the time of diagnosis (13 dogs) or lost to follow‐up (10 dogs). There were 90 cases for analysis. The survival times were compared based on the primary tumor site, site of metastasis, treatment given, age, sex and breed.

Results:  Survival times in days ranged from 0 to 1583 days. The overall median survival time was 76 days. The one‐year, two‐year and three‐year percent survival were 7%, 4.7% and 3.5%, respectively. Treatment included various combinations of chemotherapy, surgery, radiation therapy, bisphosphonates and NSAIDs. Findings included an increased survival time with surgery and adjuvant therapy compared with surgery alone and a decreased survival time with metastasis to the lung or lymph node.

Conclusions:  Treatment of dogs with stage III osteosarcoma can result in variable survival times. Multimodality therapy appears to result in longer survival times. Metastasis to the lung or lymph node correlated with a decreased survival time.
Long‐Term Totally Implantable Venous Access Ports in Dogs and Cats Receiving Chemotherapy
Authors: Bryson V. , Fox L. E. , Crum H.
Published: September 2010

Introduction:  We evaluated the totally implantable subcutaneous vascular access port (VAP) in 16 cancer patients undergoing intermittent chemotherapy for more than 30 months.

Methods:  Ports were surgically placed (The CompanionPort, Norfolk Vet Products, Skokie, Illinois 60076) in the jugular vein of 12 dogs and 4 cats between 1/2002 and 7/2004. Body weight determined polyurethane catheter size (4, 5, 7 fr.). The polysulfone port, surrounded by titanium, was anchored to subcutaneous tissue in the dorsolateral neck and confirmed with C‐arm fluoroscopy. All blood samples were obtained via VAP. Nine anticancer agents, other medications, crystalloids/colloids, and whole blood were administered. Ports were flushed every 4–5 weeks with heparinized saline solution (100 IU/ml). Removed catheters were submitted for bacteriology.

Results:  Seven of 16 animals are still alive. VAP were used for 1.5 to more than 30 months with 4–60 injections/port. Catheter tips were visualized from the left atrium distally into the caudal vena cava. Adverse events included post‐operative subcutaneous bruising and/or hematoma (4/16), difficult aspiration (4/16), catheter malposition (1/16), positional flushing (1/16), and occlusion requiring replacement (1/16). No thrombus formation or extravasation was evident. Bacterial colonization without signs of septicemia was observed in 3/4 catheters.

Conclusions:  VAP are an effective way of achieving long‐term venous access in the dog and cat. Complications are typically minor and infrequent.
Magnetic resonance imaging characterization of canine splenic lesions
Authors: Clifford C. A. , Pretorius E. S. , Weisse C. , Siegelman E. S. , Sorenmo K. U. , Skorupski K. , Drobatz K. , Burgess K. E. , Fred R. M. , Glass E. N. , Murray M. , Solomon J. R.
Published: September 2010

Introduction:  Splenic lesions are a common finding in veterinary medicine and typically 1/2 to 2/3 of these lesions are malignant. Due to the limited accuracy of ultrasound, unnecessary exploratory surgeries/biopsies may be performed for benign lesions and treatment may be delayed for malignant ones. Splenic lesions are rare in people. MR imaging, with its inherently high soft tissue contrast, is efficacious in imaging the human spleen. We have previously demonstrated the efficacy of MRI to differentiate canine hepatic lesions. In that study 8 splenic lesions were all accurately characterized. This current study represents a further evaluation of splenic lesions.

Methods:  In this prospective study, 27 dogs with splenic lesions were accrued. Histopathological/cytological confirmation of lesions occurred either before or shortly after imaging. MRI clinicians were blinded to histopathology results. MR (General Electric, 1.5 Tesla) images using a variety of sequences were obtained before and after intravenous administration of gadolinium.

Results:  32 lesions (9 malignant, 23 benign) were evaluated in 27 dogs. Lesions were confirmed via histopathology (n = 20) or cytology (n = 12). Benign lesions included, EMH (n = 7), hematoma/hemorrhage (n = 5), lymphoid hyperplasia (n = 9), and hemangioma (n = 2). Malignant lesions included anaplastic sarcoma (n = 3), malignant histiocytosis (n = 2), hemangiosarcoma (n = 2), plasma cell tumor (n = 1) and adenocarcinoma (n = 1). The overall accuracy in differentiating benign from malignant lesions was 88%(29/32 lesions). The overall sensitivity and specificity were 100%(95% CI, 66–100) and 87%(95% CI 66–97).

Conclusions:  Based upon these results, MRI is both sensitive and specific in distinguishing between malignant and benign splenic lesions.
Coarse Fractionated Radiation Therapy for Pituitary Tumours in Cats: A Retrospective Study of 10 Cases
Authors: Brearley M. J. , Polton G. A.
Published: September 2010

Introduction:  Pituitary tumours are uncommon in cats. Signs may be due to either an expansile mass or paraneoplastic effects (acromegaly and/or unstable diabetes mellitus). There are a few small case series providing evidence of efficacy for radiotherapy of pituitary tumours in cats. This retrospective study describes the outcome of ten cats with pituitary tumours treated with course‐fractionated radiation.

Methods:  The medical records of cats with MRI‐confirmed pituitary tumours that underwent radiotherapy were reviewed. A standard coarse‐fractioned radiation protocol was used; 37 Gy in 5 once‐weekly fractions using two parallel‐opposed 4MeV X‐ray beams. Survival times were calculated from date of first radiation dose.

Results:  Ten cats with pituitary tumours underwent radiotherapy. 5 cats had CNS signs and 5 had evidence of growth hormone excess (1 cat also showed CNS signs). 2 cats with pre‐existing moderate to severe CNS signs died of unknown causes before completing the radiation course. Of the remaining 4 with CNS signs, 3 had complete resolution of signs and the fourth showed partial improvement. Of the 5 cats with unstable diabetes, 2 no longer required insulin and 3 became stable at a lowered dose. The median survival time was 77.6 weeks. 6 cats died: 2 without completing the radiation course, 2 from unrelated causes (CRF, VAFS) and 2 from relapse and/or progression of CNS signs. 4 cats remain alive (range 34–191 weeks).

Conclusions:  Radiation therapy is confirmed as an effective treatment for pituitary tumours in cats giving extended survival and control of both direct mass effect and paraneoplastic signs.
Prostatic Neoplasia: In Search of a Treatment
Authors: Henderson R. A. , Smith A. N. , Higginbotham M. L.
Published: September 2010

Introduction:  Complete prostatectomy with healing and normal urinary function is reported for some prostatic diseases. However incontinence rates of up to 100% for neoplasia‐affected dogs have dampened surgeons’ enthusiasm for performing prostatectomies. Hypothesizing that incontinence following prostatectomy for prostatic cancer is related to extensive dissection associated with advanced invasion, it is proposed that there exists a subset of dogs with prostatic cancer that require minimal dissection and have the potential for quality survival for pet and owner.

Materials and Methods:  Dogs that received prostatectomy for neoplasia were qualitatively reviewed for commonalities associated with an outcome viewed as successful by owner and veterinarian.

Results:  Early diagnosis (prostatomegaly‐castrated population, high index of suspicion, ultrasound guided cytology) with referral center staging (ultrasound, contrast CT) enabled selection of appropriate surgery (urinary drainage, stent, prostatic enucleation, extrapelvic urethral anastomosis, cystoprostatourethrectomy with ureterocolonic anastomosis) for several dogs.

Conclusions:  A massive demographic shift toward castrated dogs and widespread availability of abdominal u/s can distinguish dogs that may benefit from advanced staging technology and treatment for locally confined prostatic neoplasia.

Application:  Step one is local control. Though based on a dribble of cases, it is clear that some dogs with prostatic cancer can be cured and live high quality lives. We wanted to leak these results so that oncologists will encourage early aggressive investigation and referral for prostatomegaly in castrated dogs.
Increase in Regulatory T Cells in Dogs with Cancer Undergoing Chemotherapy
Authors: Biller B. J. , Walter C. U. , Lana S. E. , Dow S. W.
Published: September 2010

Introduction:  Regulatory T cells (Treg) are a naturally occurring population of T cells phenotypically identified by co‐expression of CD4 and the IL‐2 receptor (CD25). Theyplay a critical role in the control of tolerance and autoimmunity and have also been implicated in impairment of anti‐tumor responses. We hypothesized that levels of Treg would be higher in cancer‐bearing dogs than in normal dogs and that they would decrease with chemotherapy.

Methods:  Serial PBMC were isolated from twenty cancer‐bearing dogs receiving either single‐agent doxorubicin or the Madison‐Wisconsin protocol. The following time points were studied: pre‐treatment, day 2, week 1, week 3, 3 months and 6 months after initial treatment. Ten age‐matched, normal dogs were also studied. PBMC were immunostained with directly conjugated antibodies to CD4, CD8, CD44 and IL‐2 receptor and then evaluated by flow cytometry.

Results:  Low numbers of lymphocytes with the CD4+/IL‐2R+ phenotype were detectable in both normal and cancer‐bearing dogs. A statistically significant increase in the percentage of IL2‐R+/CD4+ T cells was observed in the cancer‐bearing dogs beginning two days after chemotherapy and persisting throughout treatment. The percentage of IL‐2R+/CD4+ T cells was also increased in pre‐treated cancer‐bearing dogs compared to control dogs.

Conclusion:  The percentage of IL‐2R+/CD4+ T cells was generally higher in dogs with cancer than in healthy dogs. Unexpectedly, the percentage of IL‐2R+/CD4+ cells increased during chemotherapy which suggests that chemotherapy may exert immunosuppressive effects through a previously undescribed mechanism. The identity of these CD4+/IL‐2R+ T cells as true Treg awaits additional characterization studies.
Evaluation of Intracavitary Chemotherapy Delivery for the Treatment of Mammary Carcinoma
Authors: De Lille A. , Rizzo S. A. , Hudacheck S. F. , Chubb L. S. , Kwong G. , Dernell W. S.
Published: September 2010

Introduction:  The purpose of this study was to evaluate tolerability and efficacy of local (intracavitary) delivery of paclitaxel from a gel polymer (poloxamer 407) following marginal (histologically incomplete) resection of mammary carcinoma in a mouse model

Methods:  In vitro sensitivity to paclitaxel as well as poloxamer‐taxol mixture (polotax) was determined for 4 human breast tumor cell lines using the MTS‐assay (Promega). Nude mice were then injected with MCF‐7/ADR (AdriamycinR resistant) cells. Tumor growth was monitored by imaging of luciferase activity with a CCD camera (IVIS system, Xenogen). Primary tumors were allowed to grow to 3 different size ranges. At the time of primary tumor resection, animals were randomly assigned to treatment groups comparing intracavitary (in the wound) polotax to intravenous paclitaxel. Mice were imaged weekly to evaluate tumor regrowth and metastasis.

Results:  All cells lines demonstrated sensitivity to paclitaxel and three of the four cell lines demonstrated improved cytotoxicity with polotax compared to drug delivered alone. One of 9 mice treated with polotax had local regrowth (by 60 days) compared to 6 of 9 mice treated with intravenous paclitaxel. One of 9 mice treated with polotax had distant metastasis at 60 days compared to 5 of 8 mice treated with intravenous paclitaxel. These effects were seen with tumors of all sizes.

Discussion/Conclusions:  Poloxamer delivery of paclitaxel appears to result in improved efficacy compared to paclitaxel alone. Improved local and systemic control of mammary carcinoma is seen following intracavitary poloxamer delivery of paclitaxel compared to paclitaxel alone in this mouse model.
Anti‐Tumor Effect of Adenovirus‐Mediated P53 Gene Therapy on the Growth of Canine Osteosarcoma Transplanted into Nude Mice
Authors: Kanaya N. , Yazawa M. , Mochizuki M. , Nishimura R. , Sasaki N. , Setoguchi A. , Ohno K. , Tsujimoto H.
Published: September 2010

Introduction:  It has been reported that 40–50% of canine osteosarcoma cases have p53 mutations. The p53 tumor supressor gene plays a central role in cell cycle regulation and induction of apoptosis. We previously showed that adenoviral vector expressing canine P53 (AxCA‐cp53) inhibited growth of cultured canine osteosarcoma cell lines. Here, we evaluated anti‐tumor effect of adenovirus‐mediated p53 gene therapy on the growth of canine osteosarcomas transplanted into nude mice.

Methods:  Nine nude mice were subcutaneously injected with cells of a canine osteosarcoma cell line (POS) having p53 gene mutation. The transplanted tumors formed into nude mice were injected with AxCA‐cp53, AxCA‐LacZ (adenovirus vector expressing LacZ) or PBS (3 mice each) 7 times during 15 days. Tumor sizes were measured every 3 days for 27 days after injection with the adenovirus vectors. Expression efficiency of the adenovirus‐mediated gene transfer was examined by X‐gal staining and P53 immunostaining. Effects of the P53 expression on cell cycle control were examined by RT‐PCR for expression of p21 gene downstream of P53.

Results:  Significant differences in the tumor size was observed between the transplanted osteosarcoma tissues injected with AxCA‐cp53 and those injected with AxCA‐LacZ or PBS. Expressions of LacZ and P53 were confirmed at the injection sites of the tumors. Moreover, p21 mRNA expression was shown to be induced in the AxCA‐cp53‐injected tumors, indicating the funciton of P53 to induce cell cycle arrest.

Conclusions:  Adenoviral vector expressing canine P53 inhibited the growth of canine ostersarcoma transplanted into nude mice.
Histological Subtypes of Canine Lymphoma are Characterized by Unique Gene Expression Profiles
Authors: Kisseberth W. C. , Kosarek C. E. , Auer H. , Kornacker K. , Valli V. E. , Couto C. G.
Published: September 2010

Introduction:  In people, specific histological subtypes of lymphoma have different biological behaviors. Similar differences are recognized for some subtypes of canine lymphoma. Recently, microarray expression analysis has been used to identify unique gene expression profiles that characterize specific subtypes of human lymphoma. In this study, we use high‐density oligonucleotide microarrays to classify canine lymphomas based upon their gene expression profiles.

Methods:  Total RNA was isolated from 27 lymphoma and 3 normal lymph node canine biopsy samples. Microarray analysis was performed as described (http://www.dnaarrays.org). cRNA was hybridized to GeneChip® Canine Genome Arrays (Affymetrix), that contain over 21,700 probe sets, measuring transcripts across the entire canine genome. Gene expression levels were estimated from GeneChip® PM probe intensities by means of an enhanced version of the Li‐Wong PM‐only algorithm. Exceptional micro‐clusters of gene expression profiles were identified by means of a custom “agglomerative partitioning” algorithm which recursively partitions sets of gene expression profiles into a hierarchy (KTree) of “typical”(largest) and “exceptional”(smaller) subsets.

Results:  We identified microclusters of gene expression profiles that distinguish normal lymph node, B‐cell lymphoma, T‐cell lymphoma, and diffuse large B‐cell lymphoma phenotypes. We confirmed that, when standard hierarchical cluster analysis is applied to the identified gene expression profiles, tumors consistently cluster in concordance with their histological diagnosis.

Conclusions:  Histologically defined subtypes of canine lymphoma are characterized by distinct gene expression profiles. Gene expression profiling has great potential for the identification and verification of histological subtypes of lymphoma and for increasing understanding of the molecular pathology of canine lymphoma.
Antitumor Activity of Mycobacterial Cell Wall‐DNA Complex (MCC) Against Canine Urinary Bladder Transitional Cell Carcinoma Cells
Authors: Knapp D. W. , Filion B. , Filion M. C. , Phillips N. C.
Published: September 2010

Introduction:  Mycobacterial cell wall‐DNA complex (MCC) is a bifunctional anticancer agent that induces cancer cell apoptosis and stimulates cytokine synthesis by immune cells. Intravesical MCC is currently being evaluated in humans with high‐grade urinary bladder cancer. Evaluation of MCC in dogs with transitional cell carcinoma (TCC) will allow mechanistic studies in a natural animal model of TCC, and a potentially beneficial therapy for dogs with this cancer. In this study, we have determined the anticancer activity of MCC against canine TCC cells in vitro.

Methods:  Canine TCC cells (K9TCC cell line) were incubated with MCC (0.05–100 μg/ml, 0.5–72 hours). Cellular proliferation was measured by MTT reduction. Cell cycle was analyzed by flow cytometry with propidium iodide. Apoptosis was identified by flow cytometry using anti‐active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies. Apoptosis‐inducing activity of 100 μg/ml MCC in combination with piroxicam (0.1–1.0 uM) was evaluated.

Results:  MCC inhibited K9TCC cell proliferation in a concentration‐dependent manner (maximal activity – 45% at 100 μg/ml MCC) in association with the presence of activated caspase‐3 and cleaved PARP. Inhibition of proliferation and apoptosis‐inducing activities of MCC were independent of cell cycle phase. A thirty‐minute exposure of MCC was sufficient for optimal activity. Piroxicam (0.5 uM) enhanced apoptosis‐inducing activity of MCC.

Conclusions:  MCC induces apoptosis in K9TCC cells. This activity is potentiated by piroxicam. Following positive results in vitro, in vivo studies have been initiated. One dog, treated to date, has had a minor reduction in tumor volume following the first course of treatment with no treatment‐related toxicity.
Efficacy of C/B‐OPP rescue for the treatment of relapsed canine lymphoma (1998–2004)
Authors: LeBlanc A. K. , Mauldin G. E. , Milner R. J. , Cooke K. L. , LaDue T. A. , Mauldin G. N.
Published: September 2010

Introduction:  MOPP chemotherapy is useful for relapsed canine lymphoma. This study evaluates the efficacy of this protocol after substitution of CCNU (lomustine) or BiCNU (carmustine) for mechlorethamine (C/B‐OPP).

Methods:  Patient signalment, response to chemotherapy, toxicity and survival data were abstracted from medical records of dogs from receiving C/B‐OPP between 1998 and 2004.

Results:  Fifty‐eight dogs received C/B‐OPP rescue chemotherapy during the study period. The median remission duration after initial chemotherapy, consisting of CHOP‐based therapy in 91% of dogs, was 133 days (range, 10 to 932 days). Thirty‐eight of fifty‐eight dogs (66%) responded to C/B‐OPP rescue after relapse (22 CR, 16 PR), for a median of 48 days (range, 2 to 359 days). Overall, C/B‐OPP extended survival by a median of 90 days (range, 2 to 426 days). Twenty‐four dogs (41%) experienced one or more episodes of Grade II or higher gastrointestinal toxicity. Forty‐one dogs (71%) experienced one or more episodes of Grade II or higher hematologic toxicity. Twelve dogs (20%) developed regenerative anemia with diarrhea consistent with gastrointestinal hemorrhage. Treatment delays due to hematologic toxicity occurred in 37 dogs (63%). There were 16 nonfatal treatment‐related episodes of sepsis requiring hospitalization. 5 dogs died due to sepsis and/or chemotherapy‐related complications.

Conclusions:  C/B‐OPP chemotherapy has activity against relapsed canine lymphoma which is similar to that of traditional MOPP rescue therapy. Moderate to severe hematologic toxicity was observed. Further work is warranted to optimize drug doses and scheduling.
Characterization, Expression and Function of c‐MET in Canine Spontaneous Cancers
Authors: Liao A. T. , McMahon M. , London C. A.
Published: September 2010

Introduction:  Aberrant expression of the proto‐oncogene c‐Met has been noted in a variety of human cancers. In dogs, inappropriate Met expression has been identified in canine osteosarcoma (OSA) tumor samples. To better define the potential role of Met dysregulation in canine cancer, we cloned canine Met, HGF, and HGF activator and evaluated their expression patterns in a variety of canine tumor cell lines.

Methods:  Canine Met, HGF, and HGF activator were cloned from normal canine liver and canine OSA cell lines using primers based on regions of homology between mouse and human sequences as well as 5′ and 3′ RACE.

Results:  Inappropriate expression of Met was found in canine cell lines derived from OSAs, mast cell tumors, histiocytic sarcomas, hemangiosarcoma, and melanomas. Both HGF and HGF activator were found to be expressed in several of these tumor cell lines, providing evidence of a possible autocrine loop of Met stimulation. Incubation of canine tumor cell lines with rhHGF resulted in Met autophosphorylation and activation of the downstream signaling elements Gab1, Akt and Erk1/2. Scattering of tumor cells in response to HGF occurred under conditions of cell stress, such as serum starvation. Lastly, the Met inhibitor PHA‐665752 blocked HGF induced phosphorylation of canine Met and Gab1.

Conclusions:  These studies provide evidence that similar to the case in human tumors, aberrant Met expression may play an important role in the biology of canine cancer. As such, inhibition of Met function may represent a potentially useful novel therapeutic approach.
Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines
Authors: Matsuura S. , Koto H. , Koshino A. , Okayama T. , Setoguchi A. , Ohno K. , Tsujimoto H.
Published: September 2010

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.

Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC50) of vincristine or doxorubicin.

Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC50 of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.

Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.
Characterization of the Biological Behavior of Appendicular Osteosarcoma in Rottweilers with a Comparison to Other Breeds
Authors: McNeill C. J. , Clifford C. A. , Shofer F. S. , Samluk M. R. , Van Winkle T. J. , Sorenmo K. U.
Published: September 2010

Introduction:  The clinical impression of many oncologists is that Rottweilers with appendicular osteosarcoma (OSA) have an unfavorable prognosis despite standard therapy (amputation and chemotherapy). The purpose of this study was to compare clinical characteristics, biological behavior and outcome of OSA in Rottweilers to that of other breeds.

Methods:  107 OSA cases were studied: 56 clinical cases (17 Rottweilers) and 54 necropsy submissions (13 Rottweilers) of OSA (3 cases overlapping). Information regarding signalment, prognostic factors, treatment and outcome was obtained from medical records and follow‐up with owners. Data were analyzed by chi‐square test, t‐test, and log rank test.

Preliminary Results:  Compared to other breeds Rottweilers were more likely to have femur (23% vs. 16%) and ulna (15% vs. 3%) and less likely to have tibia (8% vs. 27%) as their primary tumor location (p = 0.04). In the necropsy group, Rottweilers had a younger age at death (6.4 vs. 10 years, p = 0.0002) and a greater body weight at diagnosis (49 vs. 37 kg, p = 0.002). No statistical differences in the age at diagnosis or survival time were noted in Rottweilers vs. other breeds. Metastasis to lymph nodes (31% vs. 10%, p = 0.08) and lungs (64% vs. 49%, p = 0.24) was more frequent in Rottweilers. There was no difference in chemotherapy drugs given; however, Rottweilers were 20 times more likely to receive NSAIDs (p = 0.0004).

Conclusion:  Greater body weight with a predilection for certain tumor locations may contribute to OSA development in Rottweilers. A younger age at death may suggest a more aggressive phenotype.
Expression of E‐cadherin in Canine Anal Sac Apocrine Gland Adenocarcinoma and its Association with Survival
Authors: Polton G. A. , Adams V. , Green L. M. , Brearley M. J. , Scase T. J.
Published: September 2010

Introduction:  Inverse associations have been shown between E‐cadherin expression and degree of differentiation in canine mammary tumours and with the acquisition of malignancy in human epithelial cancers. The purpose of this study was to examine whether there was an association between prognosis, and the distribution or intensity of immunohistochemical staining for E‐cadherin in anal sac adenocarcinoma.

Methods:  Formalin‐fixed paraffin‐wax embedded canine anal sac adenocarcinoma specimens were obtained for 36 patients for whom clinical data, including survival statistics, were available. Canine mammary adenoma tissue was employed as the positive and negative controls with antibody diluent replacing the primary antibody in the latter. Samples were scored according to proportion of cells showing positive cytoplasmic membranous staining, intensity of cytoplasmic membranous staining and proportion of nuclei staining. As quality control, a sample of 10 slides was randomly selected for repeat evaluation; results exactly matched those obtained in the first assessment.

Results:  All anal sac adenocarcinoma cells expressed E‐cadherin to varying degrees. There was no evidence of an association between survival time and the intensity of staining or the proportion of nuclei staining. Patients whose tumours exhibited >75% positive cytoplasmic membranous staining survived significantly longer than those exhibiting <75% staining with median survival times of 1100 and 479 days respectively (log rank test: χ2 = 3.89, 1 DF, p = 0.049).

Conclusion:  Immunohistochemical evaluation of the proportion of cells in anal sac adenocarcinoma samples exhibiting positive cytoplasmic membranous staining for E‐cadherin may allow improved determination of prognoses and could aid clinical decision making.
The effect of deracoxib and piroxicam on viability of canine osteosarcoma cells in vitro
Authors: Royals S. , Farese J. , Milner R. , Lee‐Ambrose L.
Published: September 2010

Introduction:  Cyclooxygenase‐2 (COX‐2) inhibitors are being used increasingly in cancer therapy. Although the effects of COX‐2 inhibitors have been evaluated extensively in carcinomas, less is known about their effects in sarcomas. Since the majority of dogs with appendicular osteosarcoma (OSA) are treated for pain with a non‐steroidal anti‐inflammatory drug (some COX‐2 selective) prior to definitive treatment, it is important to determine the effects that commonly used NSAIDS have on tumor cell growth.

Methods:  Established canine osteosarcoma (POS, HMPOS and COS31) and canine fibroblast cell lines were maintained in culture under standard conditions. Cells were incubated with either deracoxib (1 uM to 500 uM) or piroxicam (1 uM to 1000 uM). Cell viability was assessed at 72 hours by cell counts and the MTT assay. The DNA fragmentation analysis was utilized to assess for apoptosis induction.

Results:  Deracoxib concentrations ≥100 uM and piroxicam concentrations ≥500 uM significantly reduced mean cell viability of all three OSA cell lines (lowest cell viability percentages 20% and 32%, respectively). Deracoxib concentrations ≥250 uM and piroxicam concentrations ≥500 uM also reduced viability of fibroblasts; however, the cell viability percent was reduced to only 54% and 68%, respectively, of the control value. Exposure of OSA cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation.

Conclusions:  Deracoxib and piroxicam demonstrated a cytotoxic effect on canine osteosarcoma cells. There was no evidence of apoptosis induction at the concentrations evaluated. Further investigation will need to be performed to determine whether either drug exhibits anti‐tumor effects in vivo.
Serum and Plasma Vascular Endothelial Growth Factor Levels in Various Cat Populations
Authors: Smith Annette N. , Vaughn J. A. , Whatley M. , Truett A. , Keen J.
Published: September 2010

Introduction:  Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. Elevated levels within tumors, and in serum, plasma, and tumor effusion have been correlated with the development of metastatic disease, recurrence, and poor prognosis in many tumors in humans. Canine VEGF has been sequenced as homologous with the human form, and elevated serum and plasma VEGF have been found in dogs with hemangiosarcoma. Feline VEGF has also been sequenced, and shares homology with the human and canine forms.

Materials and Methods:  Stored serum and plasma samples from normal cats, cats with various neoplasms, and cats with non‐neoplastic disease were evaluated with a commercial ELISA kit (R&D Systems, Minneapolis MN). Samples were run in duplicate, and a standard curve was performed for each plate. The data were analyzed for differences between populations, and between serum and plasma levels in the same patient to determine the optimal sample for evaluating VEGF in cats.

Results:  In seven apparently healthy cats mean plasma VEGF was 95.6 pg/mL. In non‐neoplastic disease (7 cases), mean plasma VEGF was 117.3 pg/mL and mean serum VEGF level was 219.7 pg/mL. In ten tumor‐bearing patients mean plasma VEGF was 247.1 pg/mL, and mean serum VEGF was 322.3 pg/mL. Statistical analysis showed no significant difference between mean serum and plasma VEGF concentrations within each group or between groups (p > 0.05).

Discussion:  Serum and plasma VEGF levels could not be used to distinguish between healthy cats and cats with neoplastic or non‐neoplastic disease.
Efficacy and Toxicity of Intracavitary Administration of Pegylated Liposomal Encapsulated Doxorubicin (DOXIL) in Dogs with Hemangiosarcoma
Authors: Sorenmo Karin , Samluk Marissa , Clifford Craig , Baez Jennifer , Overley Beth , Skorupski Katherine , Oberthaler Karen , Barrett Jeffrey , Winkle Tom Van , Seiler Gabriela , Poppenga Robert , Shofer Frances
Published: September 2010

Introduction:  Canine hemangiosarcoma (HSA) is a fatal malignancy and most dogs die within 6–8 months of diagnosis. The spleen is a common primary site, representing 50% of all cases. These dogs typically present with clinical signs due to tumor rupture and intra‐abdominal dissemination; the abdomen is also the main site of disease progression when these patients fail. Direct delivery of chemotherapy into the abdominal cavity may therefore be a rational approach in this malignancy.

Methods:  14 dogs with stage 2 or 3 splenic HSA were recruited. Doxil at a dose of 1 mg/kg was diluted in saline and administered via ultrasound‐guidance into the abdominal cavity. The dogs were scheduled to receive 4 treatments every 3 weeks. Samples of plasma and abdominal fluid were collected for pharmacokinetic analysis. All dogs were monitored for recurrence and complete necropsies were requested at death.

Results:  8 dogs with stage 3 and 6 dogs with stage 2 HSA were enrolled. All 14 dogs have died, 12/14 due to tumor and 2 from other causes. There was no difference in median survival days between stages (stage 2: 244, stage 3: 125, p = .22). All 12 dogs that died due to tumor‐related causes failed with intra‐abdominal recurrence. Necropsies showed that the dogs in this study had relatively fewer extra‐abdominal metastasis compared to dogs treated with systemic chemotherapy. Pk analysis showed detectable plasma doxorubicin 1 and 2 weeks after treatment.

Conclusion:  Direct abdominal administration of Doxil did not prevent intra‐abdominal recurrence; however, it appeared to provide effective systemic coverage.