Measurement of telomerase activity in dog tumors

Telomeres are specific structures present at the end of liner chromosomes. DNA polymerase can not synthesize the end of liner DNA and, as a result, the telomeres become progressively shortened by successive cell divisions. To overcome the end replication problem, telomerase adds new telomeric sequences to the end of chromosomal DNA. The enzyme activity is undetectable in most normal human adult somatic cells, in which shortening of the telomere is thought to limit the somatic-cell life span. In contrast to normal somatic cells, many human tumors possess telomerase activity. The present study looked at whether telomerase activity might serve as a marker for canine tumors. Telomerase activity was measured using the telomeric repeat amplification protocol assay. Normal dog somatic tissues showed little or no telomerase activity, while normal testis exhibited a high level of telomerase activity. We measured telomerase activity in tumor samples from 45 dogs; 21 mammary gland tumors, 16 tumors developed in the skin and oral cavity, 7 vascular tumors and 1 Sertoli cell tumor. Greater than 95% of the tumor samples contained telomerase activity (3-924 U/2 micrograms protein). The results obtained in this study indicated that telomerase should be a useful diagnostic marker for a variety of dog tumors, and it may serve as a target for antitumor chemotherapy.     

PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

Telomerase activity in canine osteosarcoma

Appendicular osteosarcoma (OSA) is the most common primary bone tumour in dogs, and the prognosis with standard of care therapy of amputation and adjunctive chemotherapy is generally poor, with median survival times of 1 year. The ability of neoplastic cells to maintain their telomere length, by either telomerase activity or alternate methods, is an important step in tumour development and malignancy. The purpose of this study was to determine the presence of telomerase activity in canine OSA. To evaluate the frequency of alternative lengthening of telomeres in canine OSA, we have used the telomeric repeat amplification protocol in five canine cell lines and in six samples taken from clinical patients at the time of amputation. Our results reveal the presence of telomerase activity in 100% of canine OSA cell lines and 83% of clinical samples evaluated. This is in contrast to human OSA where 25–40% expression levels of telomerase are reported. Importantly, our results not only suggest that canine OSA may serve as a good model for aggressive telomerase‐positive forms of human OSA but also that antitelomerase therapy strategies for treatment of canine OSA may be more successful than in the treatment of majority of human patients with OSA.     

Telomeres and telomerase: Biological and clinical importance in dogs

In recent years in human oncology the enzyme telomerase has emerged as an ideal target for cancer therapy. This has led to the assessment of telomerase in cancers in companion animals, mainly dogs and these studies confirm that in dogs, like humans, telomere maintenance by telomerase is the primary mechanism by which cancer cells overcome their mortality and extend their lifespan. This review aims to provide an introduction to the biology of telomeres and telomerase and to discuss some of the telomere/telomerase directed therapeutic methodologies currently under development which may be of benefit to the canine cancer patient.     

Cellular proliferative and telomerase activity in canine mammary gland tumors

In canine mammary tumors, we examined the telomerase activity, proliferative activity by proliferative cell nuclear antigen (PCNA) immunohistochemistry, and percentage of apoptotic cells by the deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The relationship between these measures and histopathologic malignancy was also investigated. PCNA index was highest in malignant tumors (adenocarcinoma: 27.0%; malignant mixed tumor: 15.7%), followed by benign tumors (adenoma: 4.4%; benign mixed tumor: 5.3%), hyperplasia (2.1%), and normal mammary gland (0.9%). In adenoma and adenocarcinoma, papillary and solid types showing higher cellularity tended to have higher PCNA indices than did cystic and tubular types. Although the TUNEL index was <1% in all cases, the relationship between this measure and histopathologic diagnosis showed the same tendency as observed in PCNA immunostaining. Telomerase activity was detectable in all adenomas, benign mixed tumors, and adenocarcinomas examined. In contrast, all normal mammary glands, hyperplasias, and malignant mixed tumors were negative for telomerase. Relative telomerase activity (RTA) of adenocarcinoma (56.5) was significantly higher than that of adenoma (27.8) and benign mixed tumor (33.9), and a significant positive correlation (P < 0.001) was noted between RTA and PCNA index. No significant correlations were noted between either PCNA or TUNEL index and clinical features such as metastasis and tumor diameter. PCNA index and telomerase activity may be useful markers for judging malignancy of canine mammary tumors.     

Detection of indoleamine 2,3-dioxygenase 1-expressing cells in canine normal and tumor tissues

Cancer immunotherapy is a novel cancer treatment for canine tumors. Indoleamine 2,3-dioxygenase 1 (IDO1) is overexpressed in some human tumors and inhibits antitumor immunity. In this study, we comprehensively evaluated expression pattern of IDO1 and the nature of IDO1-expressing cells in canine normal and tumor tissues. In normal tissue samples, IDO1 expression was detected only in the lymph nodes, spleen, tonsil tissues, and colon tissues. In contrast, IDO1-positive tumor cells were observed in several tumor tissue types. This is the first study to evaluate IDO1 expression in canine normal and tumor tissues, and the results suggest that IDO1 is a promising target for novel cancer immunotherapy in dogs with tumors.     

端粒和端粒酶与肿瘤关系的研究进展

端粒是末端染色体 ,维持染色体的稳定。端粒酶是染色体末端转移酶 ,是合成端粒DNA的酶 ,对端粒的结构起着稳定的作用。端粒、端粒酶的研究始于 2 0世纪初期 ,但直到近几年科研人员才发现他们与肿瘤有着密切的关系。在人类端粒酶的研究过程中发现端粒酶在约 85 %以上的恶性肿瘤中呈阳性 ,而动物肿瘤与端粒酶的研究还是空白。文章主要介绍端粒、端粒酶的结构功能及它们与肿瘤关系的研究进展 ,并讨论世界上首例蛋鸡 J亚群禽白血病的自然发病的病例的端粒酶 TERT表达。作者曾用辣根过氧化物酶标记的链酶卵白素进行免疫组化检测 ,结果在病鸡的心、肝、脾、肺、肾、气管、十二指肠、睾丸、骨髓、脑、胸腺、法氏囊等组织中发现 TERT表达呈阳性 ,说明在蛋鸡 J亚群禽白血病发生时端粒酶活性升高 ,提示端粒酶参与了该病的发生发展机制 ,并且该结果与人类肿瘤端粒酶的活性的研究结果相一致     

Microarray analysis of differentially expressed genes of primary tumors in the canine central nervous system

The pathophysiologic similarities of many human and canine cancers support the role of the domestic dog as a model for brain tumor research. Here we report the construction of a custom canine brain-specific cDNA microarray and the analysis of gene expression patterns of several different types of canine brain tumor. The microarray contained 4000 clones from a canine brain specific cDNA library including 2161 clones that matched known genes or expressed sequence tags (ESTs) and 25 cancer-related genes. Our study included 16 brain tumors (seven meningiomas, five glial tumors, two ependymomas, and two choroid plexus papillomas) from a variety of different dog breeds. We identified several genes previously found to be differentially expressed in human brain tumors. This suggests that human and canine brain tumors share a common pathogenesis. In addition, we also found differentially expressed genes unique to either meningiomas or the glial tumors. This report represents the first global gene expression analysis of different types of canine brain tumors by cDNA microarrays and might aid in the identification of potential candidate genes involved in tumor formation and progression.     

Telomere length and telomerase activity in canine mammary gland tumors

OBJECTIVE: To measure telomere length and telomerase activity in naturally occurring canine mammary gland tumors. SAMPLE POPULATION: 27 mammary gland tumor specimens obtained during resection or necropsy and 12 mammary gland tissue specimens obtained from healthy (control) dogs. PROCEDURE: Telomere length in tissue specimens was measured by use of restriction endonuclease digestion and Southern blot analysis. Telomerase activity was measured by use of a telomeric repeat amplification protocol assay. RESULTS: Telomere length in mammary gland tumors ranged from 11.0 to 21.6 kilobase pairs (kbp; mean +/- SEM, 14.5+/-0.5 kbp) but did not differ among tumor types. Telomeres in mammary gland tumors were slightly shorter than in normal tissue specimens, but telomere length could not be directly compared between groups, because mean age of dogs was significantly different between groups. Age was negatively correlated with telomere length in control dogs but was not significantly correlated with length in affected dogs. Telomerase activity was detected in 26 of 27 mammary gland tumors and in 4 of 12 normal tissue specimens. However, telomerase activity and telomere length were not correlated in tumor specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Telomere length is maintained in canine mammary gland tumors regardless of the age of the affected dog. Measurement of telomere length may be a useful tool for monitoring the in vivo effects of telomerase inhibitors in dogs with tumors.     

Impact of telomerase status on canine osteosarcoma patients

Background: We demonstrated previously that canine osteosarcoma (OSA) cell lines and samples from clinical patients are predominantly telomerase positive. In contrast, the majority of OSA samples from human patients appear to be telomerase negative, maintaining telomere length by an alternative lengthening of telomeres (ALT) mechanism. The purpose of the current study was to examine the telomerase status of a large number of OSA samples from dogs and determine if telomerase status can serve as a prognostic factor. Hypothesis: The majority of clinical canine OSA appendicular lesions will be telomerase positive, and telomerase positivity will negatively impact disease outcome. Animals: Sixty‐seven dogs with appendicular OSA presenting to the Colorado State University Animal Cancer Center for treatment. Methods: The Telomeric Repeat Amplification Protocol was performed on tissue samples from primary canine appendicular OSA to determine the presence of telomerase activity. Telomere restriction fragment (TRF) analysis was utilized to determine telomere length and detect ALT. Outcome data were obtained in a retrospective manner and correlated with telomerase status. Results: Seventy‐three percent of canine OSA samples were telomerase positive. Telomerase status did not have an impact on disease‐free interval or survival time. Nine of 10 telomerase‐negative samples examined were consistent with an ALT phenotype, based on TRF analysis. Conclusions and Clinical Importance: These results are consistent with the hypothesis that the majority of canine OSA are telomerase positive, suggesting that telomerase may be a valuable target for canine OSA therapy. Additionally, telomerase status does not appear to be a prognostic factor in canine OSA.     

The activity of matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (TIMPs) in mammary tumors of dogs and rats

We conducted zymography for detecting the activity of matrix metalloproteinases (MMPs) and reverse zymography for the activity of tissue inhibitors of metalloproteinases (TIMPs) in canine spontaneous and rat 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor tissues. The activities of MMPs of canine mammary tumors were quite higher than those of the rat chemically induced tumors. The activities of MMPs were significantly higher in malignant tissues than in benign ones of canine tumors, whereas the activity of only MMP-2 was higher in both benign and malignant rat tumors compared to normal tissues. There were no differences of MMPs activities between benign and malignant rat tumors. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were strikingly higher in rat tumors than in canine tumors. The activities were higher in malignant tissues than in benign ones of dogs, and higher in tumor tissues than in normal mammary tissues of rats. The results of film in situ zymography for tissue localization of gelatinolytic activity showed that the digested area was more extended in malignant tumors than in benign ones of dogs. However, the area was similarly extended in both benign and malignant rat tumors. These results may indicate that the canine spontaneous malignant mammary tumors possess more aggressive nature than the rat chemically induced counterpart, resulting from the high level of MMPs and low level of TIMPs activities of the tumor tissues.     

Immunohistochemical evaluation of expression of heat shock proteins HSP70 and HSP90 in mammary gland neoplasms in bitches

Heat shock proteins have essential roles in a number of pathophysiologic conditions including carcinogenesis and represent a group of novel molecular markers in cancer management. The aim of this study was to investigate heat shock protein expression in correlation with other neoplasm traits such as: histological type, differentiation grade, proliferative activity, estrogenic receptor expression, and cyclooxygenase-2 and p53 proteins. Material for the investigation comprised 133 tumors of the mammary gland collected from bitches. In total 14 adenomas, 66 complex carcinomas, 47 simple carcinomas and 6 solid carcinomas were collected. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Expression of heat shock protein 70 was observed in all types of evaluated neoplasms. A higher average number of cells undergoing expression of heat shock protein 70, which was statistically insignificant, was established in complex and simple cancers and in cancers with the 1st and the 2nd degree of histological malignancy. Expression of heat shock protein 90 was observed in all studied neoplasms; it was very insignificant in adenomas, compared to cancers, and the highest expression was established in the solid cancers, as well as in cancers with the 2nd degree of histological malignancy. This high expression of heat shock protein 90 was correlated with proliferative activity. The results suggest that heat shock protein 90 is involved in canine mammary gland carcinogenesis. The results also suggest that heat shock protein 90 may be a prognostic factor, but this requires detailed clinical confirmation.     

Characterization, Expression and Function of c‐MET in Canine Spontaneous Cancers

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.     

Inhibition of canine telomerase in vitro and in vivo using RNAi: further development of a natural canine model for telomerase-based cancer therapies

Despite advances in cancer therapy, cancer related morbidity and mortality among humans and companion animals remains high, and there is a clear need to develop novel targeted therapies. Expression of the enzyme telomerase has emerged as a central unifying mechanism underlying the immortal phenotype of canine cancer cells and has thus become a candidate for targeted molecular therapies. In this study, the value of telomerase inhibition to target telomerase expressing cancer cells was explored using the novel mechanism of RNA interference (RNAi). Using a Lentiviral expression construct, targeting the RNA component of canine telomerase was effective at inhibiting telomerase in vitro and tumour growth in vivo, but possible resistance mechanisms are highlighted. As canine telomerase biology is more closely related to human telomerase biology than the murine system, it is proposed that this study highlights the value of natural canine models to study anti-telomerase therapies for human patients.