Deborah T. Kochevar

Doxycycline Induces Expression of P Glycoprotein in MCF-7 Breast Carcinoma Cells

ABSTRACT P-glycoprotein (P-gp) overexpression by tumor cells imparts resistance to multiple antineoplastic chemotherapeutic agents (multiple drug resistance). Treatment of tumor cells with chemotherapeutic agents such as anthracyclines, epipodophyllotoxins, and Vinca alkaloids results in induction of P-gp expression. This study was performed to determine if clinically relevant antimicrobial drugs (i.e., drugs that are used to treat bacterial infections in cancer patients) other than antineoplastic agents can induce expression of P-gp in MCF-7 breast carcinoma cells. Expression of P-gp and MDR1 mRNA was determined in samples from MCF-7 cells that were treated in culture with doxorubicin (positive control) and the antimicrobial drugs doxycycline, piperacillin, and cefoperazone. The functional status of P-gp was assessed using laser cytometry to determine intracellular doxorubicin concentrations. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to determine if the cytotoxicity of experimental drugs was related to their ability to induce P-gp expression. MCF-7 cells treated with doxycycline (MCF-7/doxy) were stimulated to overexpress P-gp, whereas cells treated with piperacillin and cefoperazone did not overexpress P-gp. MCF-7/doxy cells were compared to a positive-control subline, MCF-7/Adr, previously selected for doxorubicin resistance, and to MCF-7 cells treated with doxorubicin (MCF-7/doxo). All three sublines overexpressed P-gp and MDR1 mRNA and accumulated less intracellular doxorubicin than did control MCF-7 cells. P-gp expression was induced only by experimental drugs that were cytotoxic (doxorubicin and doxycycline). Doxycycline, a drug that has been used for treatment of bacterial infections in cancer patients, can induce functional P-gp expression in cancer cells, resulting in multidrug resistance.

Doxorubicin induced expression of P-glycoprotein in a canine osteosarcoma cell line.

Canine and human osteosarcoma are very similar with respect to clinical presentation, radiological and histopathological features, metastatic rate and pattern and response to therapy. For these reasons, canine osteosarcoma is a useful intermediate model for the disease in humans. Overexpression of P-glycoprotein, the product of the MDR1 gene, is the most important predictor of an adverse clinical course in human patients with osteosarcoma. Exposure of canine osteosarcoma cells to doxorubicin resulted in overexpression of MDR1 mRNA and P-glycoprotein. Furthermore, these cells failed to accumulate doxorubicin intracellularly and were less sensitive to vincristine-induced cytotoxicity as compared to parental cells.

Low level amplification of c-sis and c-myc in a spontaneous osteosarcoma model

Canine and human osteosarcoma are very similar clinically, radiologically and pathologically. DNA extracted from canine osteosarcomas (n = 9) and normal canine control tissues (n = 17) was examined for amplification of the c-sis, c-myc, N-myc and c-H-ras protooncogenes. Statistically significant amplification of the c-sis and c-myc protooncogenes was evident in the tumor tissues as compared to the normal control tissues (P less than 0.05). DNA and total cellular RNA from cultured canine and human osteosarcoma and fibroblast cell lines were examined for amplification or enhanced expression of c-sis and c-myc. Very low levels of c-myc and c-sis DNA amplification were noted in canine osteosarcoma cells as compared to canine fibroblasts. Immunostaining of sections of human and canine osteosarcoma for the sis gene product, PDGF B, showed similar levels and patterns of expression in both populations of tumors.

Retinoblastoma function is a better indicator of cellular phenotype in cultured breast adenocarcinoma cells than retinoblastoma expression.

Loss of or lowered retinoblastoma (Rb) expression has been included as a prognostic indicator in breast cancer. Low or no Rb expression is seen most commonly in high-grade breast adenocarcinomas, suggesting that a relationship may exist between loss of Rb and a less differentiated state, high proliferation rate, and high metastatic potential. In this study, we compared Rb function in two established breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231, and in an established immortalized mammary epithelial cell line, MCF10A. Cells were synchronized in G0/G1 and were released for several durations, at which time total Rb protein, mRNA, and Rb/E2F/DNA complex formation were evaluated. Rb protein was significantly higher in the tumor cells than in MCF10A cells. However, Rb function was high for a longer duration in MCF10A cells as compared with MCF-7 and MDA-MB-231 cells. Our data support the general conclusion that Rb function, but not necessarily Rb protein, is lower in highly malignant breast adenocarcinoma cells as compared with lower grade tumor cells. These results emphasize the relevance of assessing Rb function over Rb protein. This is particularly important if Rb is to be used as a prognostic indicator for breast adenocarcinoma.

Immunosuppressant inhibition of P-glycoprotein function is independent of drug-induced suppression of peptide-prolyl isomerase and calcineurin activity.

Purpose: P-glycoprotein is a 170-kDa plasma membrane multidrug transporter that actively exports cytotoxic substances from cells. Overexpression of P-glycoprotein by tumor cells is associated with a multidrug-resistant phenotype. Immunosuppressive agents such as cyclosporins and macrolides, have been shown to attenuate P-glycoprotein activity. However, the mechanism by which some immunosuppressants inhibit P-glycoprotein function has not been determined. Since cyclosporin and macrolide immunosuppressants inhibit calcineurin (CaN) phosphatase and FKBP12 peptide-prolyl isomerase (FKBP12 PPI) activity, studies were conducted to determine if these effects are directly related to the inhibitory effects these immunosuppressants have on P-glycoprotein function. Methods: Western blot analysis was performed to assess CaN and FKBP12 protein levels in P-glycoprotein-negative (MCF-7) and -positive (MCF-7/Adr) breast cancer cell lines. P-glycoprotein function was determined by intracellular doxorubicin accumulation and/or cytotoxicity assays before and after CaN and FKBP12 were independently inhibited by pharmacological antagonists. Results: CaN and FKBP12 levels were similar in MCF-7 and MCF-7/Adr cells. P-glycoprotein function was not affected by treatment of P-glycoprotein-expressing MCF-7/Adr cells with CaN and FKBP12 antagonists. Conclusions: These results demonstrate that the inhibitory effects of immunosuppressive agents on P-glycoprotein function are independent of CaN or FKBP12 PPI activity.

Vertically Integrated Educational Collaboration between a College of Veterinary Medicine and a Non-profit Animal Shelter

The College of Veterinary Medicine and Biomedical Sciences (CVMBS) at Texas A&M University (TAMU) has developed a multifaceted program in partnership with the Brazos Animal Shelter to provide teaching opportunities with shelter animals during all four years of the professional curriculum. In the first three semesters of the professional program, students working in small groups spend two hours per semester at the shelter performing physical examinations, administering vaccinations and anthelmintics, completing heartworm or FeLV/FIV testing, and performing simple medical treatments. In an expanded fourth-year program, groups of six students spend 16 contact hours at the shelter during two-week rotations, completing similar tasks. Through this program, each student practices animal-handling skills and routine procedures on an average of 150 to 200 dogs and cats. In addition, during third- and fourth-year surgery courses, student teams spay or neuter an average of 12 to 18 dogs or cats each week. More than 800 animals are spayed/neutered annually through this program, and each student directly participates in 12 to 15 spay/neuter survival surgeries. The program represents a creative approach to veterinary training that conscientiously uses animal resources in a positive fashion. We believe that this is a successful partnership between a state-supported veterinary college and a non-profit shelter that benefits both agencies. We encourage other veterinary colleges to explore similar partnership opportunities to provide optimal training for professional students while using animal resources efficiently.

Assessment of clinical reasoning skills in veterinary students prior to and after the clinical year of training.

OBJECTIVE To examine the effect of various clinical tracks within the veterinary medical clinical curriculum at Texas A&M University on clinical diagnostic proficiency as determined by pre- and post-training assessment. We expected that the clinical track chosen by the student would impact their measured outcome with bias toward higher scores in their chosen field. DESIGN Prospective cohort study. STUDY POPULATION 32 students from the College of Veterinary Medicine and Biomedical Sciences at Texas A&M University. PROCEDURES By use of standardized, written case scenarios, clinical reasoning was assessed twice: once prior to the clinical (fourth) year of the curriculum and again at completion of the clinical year. Students demonstrated their abilities to collect and organize appropriate clinical data (history, physical examination, and laboratory findings), determine clinical diagnoses, and formulate and implement acceptable treatment modalities. Data from clinical assessments were compared for a given cohort and correlated with other measures (eg, grades, standardized test scores, and species-specific curricular track). RESULTS Differences were detected in clinical diagnostic proficiency among students in different clinical tracks and for different species groups in the case scenarios. Tracking by species group in the clinical veterinary curriculum appeared to affect development of clinical reasoning and resulted in differential proficiency among cases for differing species groups. CONCLUSIONS AND CLINICAL RELEVANCE Differences in clinical experiences between small animal tracks and all other track opportunities (large animal, mixed animal, and alternative) influenced the development of clinical proficiency in fourth-year veterinary students during their clinical training period.

Rb LOCALIZATION AND PHOSPHORYLATION KINETICS CORRELATE WITH THE CELLULAR PHENOTYPE OF CULTURED BREAST ADENOCARCINOMA CELLS

SummaryRetinoblastoma protein (Rb) expression has been correlated with state of differentiation, proliferation rate, and metastatic potential in breast adenocarcinomas and established cell lines. These observations, based on immunoreactivity of total Rb rather than hypophosphorylated protein, do not address the relationship between functional Rb and indicators of an aggressive transformed cellular phenotype. We hypothesized that the distribution of functional Rb and the kinetics of Rb phosphorylation would differ between cell lines representing immortalized mammary epithelium (MCF10A), differentiated nommetastatic mammary adenocarcinoma (MCF-7), and poorly differentiated, highly metastatic mammary adenocarcinoma (MDA-MB-231) and that these differences would be informative of the cellular phenotype. Direct immunofluorescence microscopy was used to compare qualitatively the subcellular localization of total and hypophosphorylated Rb protein in synchronized and asynchronous cells. This technique was also used to quantitatively assess the amounts of hypophosphorylated Rb throughout the cell cycle in these representative cell lines. Total Rb stained more prominently than hypophosphorylated Rb in the nucleus of all asynchronous cells. Rb phosphorylation was more rapid in MCF-7 cells than in MCF10A cells, whereas Rb dephosphorylation appeared deregulated in MDA-MB-231 cells. We conclude that assessment of hypophosphorylated Rb may be more useful than assessment of total Rb for the evaluation of transformed breast adenocarcinoma phenotypes.

Fifty Years of Evolving Partnerships in Veterinary Medical Education

The Association of American Veterinary Medical Colleges (AAVMCs) role in the progression of academic veterinary medical education has been about building successful partnerships in the US and internationally. Membership in the association has evolved over the past 50 years, as have traditions of collaboration that strengthen veterinary medical education and the association. The AAVMC has become a source of information and a place for debate on educational trends, innovative pedagogy, and the value of a diverse learning environment. The AAVMCs relationship with the American Veterinary Medical Association Council on Education (AVMA COE), the accreditor of veterinary medical education recognized by the United Sates Department of Education (DOE), is highlighted here because of the key role that AAVMC members have played in the evolution of veterinary accreditation. The AAVMC has also been a partner in the expansion of veterinary medical education to include global health and One Health and in the engagement of international partners around shared educational opportunities and challenges. Recently, the association has reinforced its desire to be a truly international organization rather than an American organization with international members. To that end, strategic AAVMC initiatives aim to expand and connect the global community of veterinary educators to the benefit of students and the profession around the world. Tables in this article are intended to provide historical context, chronology, and an accessible way to view highlights.

A strategic approach to workforce dynamics requires collaboration and sound data

The recent release of the AVMA’s highly anticipated study of the veterinary workforce in the United States 1 was an important milestone and provided valuable empirical data for consideration. Although some believe the study vindicates their assertion that academic veterinary medicine is producing too many graduates for the current market, we suggest that the study instead indicates that the issues are more complex and demand additional scrutiny and insight. We should recognize and embrace this study for what it is: a clarifying and important step toward understanding the dynamics impacting the veterinary profession. We should also recognize that this study demonstrates a level of scientific rigor and sophistication that speaks to AVMA’s commitment to address the economic issues affecting our profession. The current operating environment for veterinary medicine has been shaped by structural economic imbalances rooted in the way owners are willing to pay for high-quality clinical care for companion animals, the high cost of medical education, Internet-era changes in the way goods and services are sold to consumers, and changes in agricultural production practices, to name just a few. These stressors have been apparent and building for many years and have been considered in a wide range of economic studies. 2 There are no easy answers for these fundamental problems, and they are each worthy of additional discussion and debate. In light of this workforce study, we would like to share some insights about academic veterinary medicine and address some misperceptions that exist regarding what colleges of veterinary medicine can and cannot do, now that excess capacity in some market sectors has been demonstrated. First, we need to remember that workforce imbalances and the economic spasms they engender periodically occur in various professions. In the early 1900s, the rise of tractors and