Stearidonic acid, a plant-based dietary fatty acid, enhances the chemosensitivity of canine lymphoid tumor cells.

Lymphoma is the most common hematopoietic tumor in dogs and humans, with similar pathogenesis and therapeutic responses. Anticancer drugs like vincristine (VCR) and doxorubicin (DOX) are often used in treating lymphoma. However, the cure rate is generally poor due to chemoresistance. Here, we sought to determine whether stearidonic acid (SDA), a plant-based dietary fatty acid, sensitizes chemoresistant canine lymphoid-tumor cells. GL-1 B-cell lymphoid-tumor cells were found to be highly sensitive to the antitumor-activity of VCR and DOX, while OSW T-cell and 17-71 B-cell lymphoid-tumor cells were moderately and fully resistant, respectively. SDA, at its non-toxic concentrations, significantly promoted the antitumor action of VCR and DOX in both OSW and 17-71 cells. SDA-mediated chemosensitization was associated with SDA inhibition of P-glycoprotein (P-gp) function. This was confirmed in HEK293 cells stably expressing P-gp as well as by increased binding-affinity of SDA to P-gp in P-gp docking analysis. SDA at its chemosensitizing concentrations did not affect the viability of healthy dog peripheral blood mononuclear cells, suggesting that SDA is non-toxic to normal dog peripheral blood leucocytes at its chemosensitizing concentrations. Our study identifies a novel dietary fatty acid that may be used as a dietary supplement in combination with chemotherapy to promote the antitumor efficacy of the chemotherapy drugs in dogs and possibly in humans with chemoresistant lymphoma.

Tumor suppressor gene p16/INK4A/CDKN2A-dependent regulation into and out of the cell cycle in a spontaneous canine model of breast cancer.

p16/INK4A/CDKN2A is an important tumor suppressor gene that arrests cell cycle in G1 phase inhibiting binding of CDK4/6 with cyclin D1, leaving the Rb tumor suppressor protein unphosphorylated and E2F bound and inactive. We hypothesized that p16 has a role in exit from cell cycle that becomes defective in cancer cells. Well characterized p16-defective canine mammary cancer cell lines (CMT28, CMT27, and CMT12), derived stably p16-transfected CMT cell clones (CMT27A, CMT27H, CMT28A, and CMT28F), and normal canine fibroblasts (NCF), were used to investigate expression of p16 after serum starvation into quiescence followed by re-feeding to induce cell cycle re-entry. The parental CMT cell lines used lack p16 expression either at the mRNA or protein expression levels, while p27 and other p16-associated proteins, including CDK4, CDK6, cyclin D1, and Rb, were expressed. We have successfully demonstrated cell cycle arrest and relatively synchronous cell cycle re-entry in parental CMT12, CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells and confirmed this by (3)H-thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. p16-transfected CMT27A and CMT27H cells exited cell cycle post-serum-starvation in contrast to parental CMT27 cells. NCF, CMT27A, and CMT28F cells expressed upregulated levels of p27 and p16 mRNA, post-serum starvation, as cells exited cell cycle and entered quiescence. Because quiescence and differentiation are associated with increased levels of p27, our data demonstrating that p16 was upregulated along with p27 during quiescence, suggests a potential role for p16 in maintaining these non-proliferative states.

Novel frameshift mutation in the p16/INK4A tumor suppressor gene in canine breast cancer alters expression from the p16/INK4A/p14ARF locus.

The INK4 family of cyclin-dependent kinase inhibitors (CKI) encode important cell cycle regulators that tightly control cell cycle during G1 to S phase. These related genes are considered tumor suppressors as loss of function contributes to the malignant phenotype. Expression of CKIs p16, p14ARF, or p15 were defective in six different canine mammary tumor (CMT) cell lines compared to normal thoracic canine fibroblasts. This suggests CKI defects are frequently responsible for neoplastic transformation in canine mammary carcinomas. p16 and p14ARF are two alternatively spliced products derived from the canine p16/INK4A/p14ARF gene locus. Despite omissions in the published p16 transcript and canine genome and the presence of GC-rich repeats, we determined the complete coding sequence of canine p16 revealing a deletion and frameshift mutation in p16 exon 1α in CMT28 cells. In addition, we determined canine p14ARF mRNA and protein sequences. Mapping of these mutations uncovered important aspects of p16 and p14ARF expression and defects in CMT28 cells shifting the p16 reading frame into p14ARF making a fusion protein that was predicted to be truncated, unstable and devoid of structural and functional integrity. This data describes an important neoplastic mechanism in the p16/INK4A/p14ARF locus in a spontaneous canine model of breast cancer.

Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial-mesenchymal Transition in Aggressive Variant Prostate Cancers.

Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (EMT)] and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin, hyaluronan synthase-3, S100 calcium binding protein A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes-mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, metronomic-topotecan single agent and combinations with DTX downregulated these EMT markers as well as CD44+ and CD44+/CD133+ "stem-like" cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single agent or in combination with DTX was potentially effective against invasive prostate cancer spread. Our RNA-seq and scRNA-seq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways. Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies.

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine.

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c(+) expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

Delivery of siRNA into breast cancer cells via phage fusion protein-targeted liposomes.

Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers a unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF-7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines. FROM THE CLINICAL EDITOR: In this study, the authors report a novel approach for targeted intracellular delivery of siRNAs into breast cancer cells through encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins.

Frequent genetic defects in the p16/INK4A tumor suppressor in canine cell models of breast cancer and melanoma.

The cyclin-dependent kinase inhibitors (CKIs) belong to a group of key cell cycle proteins that regulate important cancer drug targets such as the cyclin/CDK complexes. Gene defects in the INK4A/B CKI tumor suppressor locus are frequently associated with human cancers and we have previously identified similar defects in canine models. Many of the cancer-associated genetic alterations, known to play roles in mammary tumor development and progression, appear similar in humans and dogs. The objectives of this study were to characterize expression defects in the INK4 genes, and the encoded p16 family proteins, in spontaneous canine primary mammary tumors (CMT) as well as in canine malignant melanoma (CML) cell lines to further develop these models of spontaneous cancers. Gene expression profiles and characterization of p16 protein were performed by rtPCR assay and immunoblotting followed by an analysis of relevant sequences with bioinformatics. The INK4 gene family were expressed differentially and the genes encoding the tumor suppressor p16, p14, and p15 proteins were often identified as defective in CMT and CML cell lines. The altered expression profiles for INK4 locus encoded tumor suppressor genes was also confirmed by the identification of similar gene defects in primary canine mammary tumor biopsy specimens which were also comparable to defects found in human breast cancer. These data strongly suggest that defects identified in the INK4 locus in canine cell lines are lesions originating in spontaneous canine cancers and are not the product of selection in culture. These findings further validate canine tumor models for use in developing a clear understanding of the gene defects present and may help identify new therapeutic cancer treatments that restore these tumor suppressor pathways based on precision medicine in canine cancers.

Identification and Characterization of Key Differentially Expressed Genes Associated With Metronomic Dosing of Topotecan in Human Prostate Cancer.

Repetitive, low-dose (metronomic; METRO) drug administration of some anticancer agents can overcome drug resistance and increase drug efficacy in many cancers, but the mechanisms are not understood fully. Previously, we showed that METRO dosing of topotecan (TOPO) is more effective than conventional (CONV) dosing in aggressive human prostate cancer (PCa) cell lines and in mouse tumor xenograft models. To gain mechanistic insights into METRO-TOPO activity, in this study we determined the effect of METRO- and CONV-TOPO treatment in a panel of human PCa cell lines representing castration-sensitive/resistant, androgen receptor (+/-), and those of different ethnicity on cell growth and gene expression. Differentially expressed genes (DEGs) were identified for METRO-TOPO therapy and compared to a PCa patient cohort and The Cancer Genome Atlas (TCGA) database. The top five DEGs were SERPINB5, CDKN1A, TNF, FOS, and ANGPT1. Ingenuity Pathway Analysis predicted several upstream regulators and identified top molecular networks associated with METRO dosing, including tumor suppression, anti-proliferation, angiogenesis, invasion, metastasis, and inflammation. Further, the top DEGs were associated with increase survival of PCa patients (TCGA database), as well as ethnic differences in gene expression patterns in patients and cell lines representing African Americans (AA) and European Americans (EA). Thus, we have identified candidate pharmacogenomic biomarkers and novel pathways associated with METRO-TOPO therapy that will serve as a foundation for further investigation and validation of METRO-TOPO as a novel treatment option for prostate cancers.

The extra 16-amino-acid peptide at C-terminal NS2 of the hypervirulent type-2 bovine viral diarrhea viruses has no effect on viral replication and NS2-3 processing of type-1 virus.

Bovine viral diarrhea virus (BVDV) is an economically important cattle pathogen with worldwide distribution. Besides the segregation of noncytopathic and cytopathic (CP) biotypes, BVDV exists as two genotypes. Both genotypes cause similar disease, and the majority of type-2 BVDV (BVDV-2) is no more virulent than type-1 viruses (BVDV-1). However, some BVDV-2 viruses are hypervirulent and causative reagents of a lethal disease called severe acute bovine viral diarrhea. Amino acid (aa) sequence analysis shows that the majority of hypervirulent BVDV-2 isolates contains an extra 16 aa peptide (-SSCPVPFDPSCHCNYF-) at C-terminal NS2 region. In this study, we investigated the flexibility of the corresponding NS2 region of BVDV-1 for tolerance of this peptide insertion and its effect on viral pathogenicity. Based on an infectious cDNA clone of BVDV-1 SD-1, a cDNA clone called pASD1-IN was constructed with insertion of the 16 aa peptide in the corresponding NS2 site. In vitro transcription and transfection of Madin-Darby bovine kidney (MDBK) cells resulted in the generation of infectious chimeric virus termed ASD1-IN. ASD1-IN does not show CP effect on MDBK cells and is similar to ASD1 in viral growth. Furthermore, ASD1-IN shows an NS2-3 processing pattern similar to ASD1. These results reveal that insertion of the 16 aa peptide at C-terminal NS2 of BVDV-1, at least for SD-1, has no effect on viral replication and NS2-3 processing in MDBK cells.

Phenotype-rescue of cyclin-dependent kinase inhibitor p16/INK4A defects in a spontaneous canine cell model of breast cancer.

Mammary cancer is among the most frequently observed canine tumors in unspayed female dogs resulting in death due to metastatic disease. These tumors are excellent models of human breast cancer but until recently there was only anecdotal evidence regarding underlying genetic defects. We recently reported expression defects in the cyclin-dependent kinase p21/Cip1 and p53 among three independent canine mammary tumor (CMT) cell lines derived from spontaneous canine mammary cancers. We investigated further defects in the same three cell lines focusing on additional tumor suppressor gene defects in cyclin-dependent kinase inhibitors. p27/KIP1 appeared normally expressed and did not appear to encode inactivating mutations. In contrast, expression of p16/INK4A was defective/absent in two cell lines and normal/slightly induced in the third cell line. To determine if defects were causative in maintaining the transformed phenotype, a p16/INK4A transgene was permanently transfected followed by selection and single cell cloning. CMT/p16 clones were characterized for transgene expression, p16 protein content and phenotype including proliferation rate, cell cycle phase distribution, contact inhibition, substrate dependent cell growth and cell morphology. All cell lines appeared unique yet clear indications of phenotype rescue due to p16/INK4A transgene complementation were observed suggesting that defects in p16 expression were present in all three. In some cases cellular senescence also appeared to be induced. These data provide evidence supporting p16/INK4A mutations as causative defects promoting transformation in canine mammary cancer and further characterizes tumor suppressor gene defects with functional consequences in these cells supporting their application as spontaneous animal models of human disease.

Autologous hybrid cell fusion vaccine in a spontaneous intermediate model of breast carcinoma.

Breast cancer is among the most common malignancies affecting women and reproductively intact female dogs, resulting in death from metastatic disease if not treated effectively. To better manage the disease progression, canine mammary tumor (CMT) cells derived from malignant canine mammary cancers were fused to autologous dendritic cells (DCs) to produce living hybrid-cell fusion vaccines for canine patients diagnosed with spontaneous mammary carcinoma. The high-speed sorting of rare autologous canine patient DCs from the peripheral blood provides the autologous component of fusion vaccines, and fusion to major histocompatibility complex-unmatched CMT cells were produced at high rates. The vaccinations were delivered to each patient following a surgical resection 3 times at 3-week intervals in combination with immuno-stimulatory oligonucleotides and Gemcitabine adjunct therapy. The immunized patient animals survived 3.3-times longer (median survival 611 days) than the control patients (median survival 184 days) and also appeared to exhibit an enhanced quality of life. A comparison of vaccinated patients diagnosed with inflammatory mammary carcinoma resulted in a very short median survival (42 days), suggesting no effect of vaccination. The data showed that the development of autologous living DC-based vaccine strategies in patient animals designed to improve the management of canine mammary carcinoma can be successful and may allow an identification of the antigens that can be translatable to promote effective immunity in canine and human patients.

Bovine viral diarrhea virus is a suitable viral vector for stable expression of heterologous gene when inserted in between N(pro) and C genes.

Bovine viral diarrhea virus (BVDV) is a group of small enveloped viruses with a single-stranded, positive-oriented RNA genome of approximately 12.3 kb. BVDV genome directs the production of a viral polyprotein that is subsequently cleaved to release the mature viral proteins. To explore the potential of using BVDV as viral vector for stable expression of heterologous genes, eGFP2A was inserted in between N(pro) and C genes of a noncytopathic type-I BVDV strain SD1. eGFP2A was designed with eGFP protein in frame fused to the N terminus of the foot-and-mouth disease virus 2A protease. This strategy promised not only the correct processing of both viral N(pro) and C protein but also releasing of the chimeric protein from the nascent viral polyprotein. The recombinant reporter virus was successfully rescued in MDBK cells. In vitro study showed that eGFP2A protein, as expected, was expressed and processed properly from the nascent viral polyprotein. The reporter virus was similar to wt SD1 in viral RNA replication and protein expression and comparable to wt SD1 in growth kinetics except that this virus had a peak virus titer approximately 0.5 log(10) lower and a maximum yield about 4h later than wt SD1. In summary, these results indicated that BVDV is a suitable viral vector for stable expression of heterologous genes when inserted in between N(pro) and C genes.

An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA.

Full length cDNA clones of bovine viral diarrhea virus (BVDV) with a low-copy number plasmid backbone have not proven to be stable when propagated in bacteria. To improve stability, pBAS, a bacterial artificial chromosome (BAC) plasmid was used to construct a full length cDNA clone of BVDV strain SD1, which has a genomic size of 12.3 kb. The resulting clone pBSD1 was propagated stably for at least 10 passages in three different Escherichia coli strains at two different incubation temperatures as determined by sequencing the progeny plasmids. In vitro transcripts derived from pBSD1 were homologous in size and had an infectious efficiency as high as approximately 5.0 x 10(5)FFU/microg RNA in MDBK cells. The recovered virus, BSD1, harbored the five artificially introduced silent point mutations as genetic markers and was similar to wild type SD1 in viral growth kinetics, RNA replication, and protein expression. This BAC clone provides a stable reverse genetics system for manipulation and study of BVDV genes.

Generation and characterization of an Npro-disrupted marker bovine viral diarrhea virus derived from a BAC cDNA.

In vitro studies showed that N(pro) protein of bovine viral diarrhea virus (BVDV) interferes with cellular antiviral defense. To understand the role of N(pro) protein in successful viral invasion of the host and establishment of the lifetime persistence, an infectious N(pro)-disrupted virus with a noncytopathic (NCP) background is desired. In this study, an N(pro)-disrupted cDNA, pBSD1-N(pro)/eGFP2A, was constructed based on an infectious full-length BAC cDNA clone of NCP BVDV strain SD1, pBSD1. In this clone, whole N(pro) gene except its first 57 nucleotides (nt) was in frame substituted with an eGFP2A sequence. eGFP2A was constructed by in frame fusing a foot-and-mouth disease virus 2A protease (FMDV 2A(pro)) to C-terminus of eGFP. Intramolecular cleavage of FMDV 2A(pro) at its C-terminal glycine-proline dipeptide will release the viral nucleocapsid protein from the nascent viral polyprotein and the processed eGFP2A protein will then act as a marker protein. The resulting BAC cDNA clone was propagated stably for at least 10 passages in E. coli strain XL1-blue as determined by sequencing the progeny plasmids. The rescued virus, BSD1-N(pro)/eGFP2A, showed a peak virus titer approximately 1.2 log(10) lower and a maximum virus yield about 20 hr later than wt SD1, respectively, and was similar to wt SD1 in viral RNA replication and protein expression. FACS, fluorescent microscopy and western blotting assays confirmed that functional eGFP2A protein was expressed and processed properly in MDBK cells. In summary, the availability of BSD1-N(pro)/eGFP2A with a stable viral genome would facilitate the investigation of the role of N(pro) protein in transplacental transfer of BVDV and establishment of persistent infection in bovine fetus.

An allogeneic hybrid-cell fusion vaccine against canine mammary cancer.

Mammary cancer is among the most prevalent of canine tumors frequently resulting in death due to metastatic disease. Most tumors fail to raise an effective immune reaction making improving immune recognition a priority. Hybrid-cell fusion strategies have been employed to load dendritic cell populations with tumor cell antigens to stimulate immune recognition; however, recovery, heterogeneity and quality of primary cells from patients present enormous challenges. We employed allogeneic cell lines to develop an improved hybrid-cell fusion strategy and evaluated immune reactions in normal laboratory beagles. Such a strategy relies on enhanced immune recognition of allogeneic tumor cell antigens by antigen presenting cells. Optimized PEG-promoted fusions between uniquely stained canine mammary tumor CMT12 or CMT28 cells and a dendritic cell-like DH82 cell fusion partner resulted in greater than 40% hybrid-cell fusion populations by flow cytometry and fluorescence microscopy. Hybrid-cell fusions were delivered by direct ultrasound guided injection into popliteal lymph nodes of laboratory beagles. Only hybrid-cell fusions provided statistically significant enhancement of cell-mediated immunity ((51)Cr-release assay) compared to innate reactions in naïve vehicle injected dogs while dogs vaccinated with either single cell component alone did not. Vaccination with hybrid-cell fusions enhanced IFN-gamma expression in sorted CD8+ and CD4+ cells but not in CD4-/CD8- cells consistent with a CTL response. Cell-mediated immune assays revealed strong reactions against matched (vaccine component) CMT cells and unmatched CMT cells indicative of an immune response to mammary cancer antigens common to both cell lines. These results provide proof of principle for development of an allogeneic vaccination strategy against canine mammary cancer.

Evaluation of 14-3-3 sigma as a potential partner of p16 in quiescence and differentiation.

p16 is an important tumor suppressor gene encoded by the INK4A/ARF/INK4B gene locus that is conserved in humans, rodents, and canids. p16 regulates cell cycle in early G1 phase inhibiting transition out of cell cycle from G1/S phase by regulating a multi-protein control complex. p16-associated proteins, cyclin D, CDK4, and CDK6, experience expression level decreases or do not change during cell differentiation and quiescence in contrast to constant p16 expression in post-proliferative cell phases. We hypothesized that p16 has alternate binding partners, other than classical proliferation-associated proteins such as CDKs, in these post-proliferative cell phases. Using co-immunoprecipitation, we have identified 14-3-3σ as a potential alternate binding partner for p16 in quiescent post-proliferative canine mammary cancer cells. Additionally, expression of 14-3-3σ was maintained as fibroblasts exit cell cycle and differentiate to adipocytes simultaneously with continued expression of p16. Based on these results, we suggest that 14-3-3σ protein may be an alternative binding partner for p16 active during cell quiescence and may associate with p16 during cell differentiation.

Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer.

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer.

Canine Mammary Carcinomas: A Comparative Analysis of Altered Gene Expression.

Breast cancer represents the second most frequent neoplasm in humans and sexually intact female dogs after lung and skin cancers, respectively. Many similar features in human and dog cancers including, spontaneous development, clinical presentation, tumor heterogeneity, disease progression and response to conventional therapies have supported development of this comparative model as an alternative to mice. The highly conserved similarities between canine and human genomes are also key to this comparative analysis, especially when compared to the murine genome. Studies with canine mammary tumor (CMT) models have shown a strong genetic correlation with their human counterparts, particularly in terms of altered expression profiles of cell cycle regulatory genes, tumor suppressor and oncogenes and also a large group of non-coding RNAs or microRNAs (miRNAs). Because CMTs are considered predictive intermediate models for human breast cancer, similarities in genetic alterations and cancer predisposition between humans and dogs have raised further interest. Many cancer-associated genetic defects critical to mammary tumor development and oncogenic determinants of metastasis have been reported and appear to be similar in both species. Comparative analysis of deregulated gene sets or cancer signaling pathways has shown that a significant proportion of orthologous genes are comparably up- or down-regulated in both human and dog breast tumors. Particularly, a group of cell cycle regulators called cyclin-dependent kinase inhibitors (CKIs) acting as potent tumor suppressors are frequently defective in CMTs. Interestingly, comparative analysis of coding sequences has also shown that these genes are highly conserved in mammals in terms of their evolutionary divergence from a common ancestor. Moreover, co-deletion and/or homozygous loss of the INK4A/ARF/INK4B (CDKN2A/B) locus, encoding three members of the CKI tumor suppressor gene families (p16/INK4A, p14ARF and p15/INK4B), in many human and dog cancers including mammary carcinomas, suggested their important conserved genetic order and localization in orthologous chromosomal regions. miRNAs, as powerful post-transcriptional regulators of most of the cancer-associated genes, have not been well evaluated to date in animal cancer models. Comprehensive expression profiles of miRNAs in CMTs have revealed their altered regulation showing a strong correlation with those found in human breast cancers. These genetic correlations between human and dog mammary cancers will greatly advance our understanding of regulatory mechanisms involving many critical cancer-associated genes that promote neoplasia and contribute to the promising development of future therapeutics.

Characterization of the CDP-like/CTAS-1 binding site in the okadaic acid response element (OARE) of the human CDK1(p34cdc2) promoter.

BACKGROUND: Transcription of CDK1 is induced at the G0/G1-phase of the cell cycle and after okadaic acid treatment and we identified the Site I okadaic acid response element (OARE -944 to -763nt) enhancer in the human CDK1 promoter. MATERIALS AND METHODS: The OARE region of the CDK1 promoter was characterized for enhancer/repressor activities. RESULTS: Transient transfection of upper and lower Site I subregions suggested enhanced transcription activity was divided between both while mobility shift assays demonstrated sequence-specific protein binding to Site IA. Site IA also formed shift complexes following serum starvation/refeeding and putative transcription factor binding sites clustered in Site IA. Oligonucleotides encoding a consensus CDP transcription factor binding site effectively competed against authentic Site IA in mobility shift assays. Mutation of the CDP-like binding sequence substantially reduced competition. DNaseI footprinting revealed binding at this site. CONCLUSION: The CDP-like recognition sequence appears to comprise the OARE binding authentic CDP and/or related factors. We termed this site the CDK1 transcription activation sequence-1 (CTAS-1) enhancer of the human CDK1 promoter.

Protective immunity induced by DNA vaccination of channel catfish with early and late transcripts of the channel catfish herpesvirus (IHV-1).

Seven full-length transcripts encoding four early and three late genes of the channel catfish virus (CCV), ictalurid herpesvirus I (IHV-1), have been cloned following rt-PCR amplification and DNA sequencing. Transcripts were selected based on their predicted association with membrane structures, identification as an envelope glycoprotein, or as a viral capsid protein. The transcripts derived from ORF 6, ORF 7, ORF 8a, ORF 10, ORF 51, ORF 53, and ORF 59 were all shown to be complete and unspliced. Each of the seven ORFs was cloned into a vaccine expression vector designed to support high levels of expression of the inserted sequence in catfish tissues. Solutions of DNA containing one each of the seven CCV ORFs, vector alone or PBS were injected intramuscularly into 4-8 cm catfish. Four to 6 weeks after injection each experimental group was challenged with one LD(50) of CCV. Single injections of DNA expression constructs containing ORF 59, encoding the envelope glycoprotein, or ORF 6, encoding a presumptive membrane protein, were found to elicit the strongest resistance to challenge compared to uninjected, PBS injected or vector injected groups. Even more effective was a combination vaccine pair in which both ORF 59 and ORF 6 expression constructs were injected. Other ORFs did not provide consistent protection to challenge above that observed in control fish. Both percent survival and kinetics of cumulative deaths were improved using the combination DNA vaccine encoding ORF 6 and ORF 59. Both ORF 6 and ORF 59 were able to elicit virus neutralizing antibodies capable of an anamnestic response on viral challenge. We believe this evidence provides adequate proof of principle for the use of DNA vaccines in channel catfish and the effectiveness of the resistance to viral infection they elicit.