Development and Evaluation of an Experiential Career Planning Assignment to Train Students to Assess Organizational Fit.

Determining if an employment opportunity will be a good match can feel daunting, especially for veterinary graduates entering the workforce. To ease this transition, veterinary educators traditionally have attempted to provide career support through interspersed didactic lectures on career options and the preparation of employment documents. While well intended, this approach fails to address the multiple dimensions of effective career planning or the reality that career planning is a lifelong endeavor. For a career planning teaching modality to be effective, it must address all stages of career planning and provide a framework that can be adapted throughout a career. Here we describe how a four-stage career-planning model, utilized throughout higher education, was employed to create a career planning assignment for guiding students in assessing organizational fit. We describe how student feedback was used to inform revisions, resulting in an improved educational experience as measured by students' perceptions of the utility of the assignment. Additional recommendations based on instructor reflection are provided to guide creation and implementation of future assignments. Given the growing support for professional skills training in veterinary medical education, we view incorporation of such learning activities as essential to preparing students to enter the modern veterinary workplace.

In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMT(P140K) vector.

OBJECTIVE: Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Lin(neg) BM) could be genetically modified with an foamy virus (FV) vector that expresses the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and selected in vivo with submyeloablative or myeloablative alkylator therapy. MATERIALS AND METHODS: Lin(neg) BM was transduced at a low multiplicity-of-infection with the FV vector, MD9-P140K, which coexpresses MGMT(P140K) and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals. RESULTS: Following submyeloablative therapy, 55% of the mice expressed MGMT(P140K) in the BM. Proviral integration was observed in approximately 50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to two integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, after delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced because BM cells from the surviving animals reconstituted secondary recipients with MGMT(P140K)-positive cells for 5 to 6 months. CONCLUSIONS: In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative than myeloablative therapy. These data indicate that a critical number of transduced stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against acute toxicity associated with myeloablative therapy.

Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents.

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

Real-Time PCR: an Effective Tool for Measuring Transduction Efficiency in Human Hematopoietic Progenitor Cells.

Accurate measurement of gene transfer into hematopoietic progenitor cells is an essential prerequisite for assessing the utility of gene therapy approaches designed to correct hematologic defects. We developed a reliable method to measure transduction efficiency at the level of the progenitor cell with real-time polymerase chain reaction (PCR) analysis of individual progenitor-derived colonies. We hypothesized that this method would demonstrate better sensitivity and specificity than are currently achievable with conventional PCR. An oncoretroviral vector containing the enhanced green fluorescent protein was used to transduce human CD34+ cells derived from bone marrow or granulocyte-colony-stimulating factor-mobilized peripheral blood. Progenitor assays were set up and colonies plucked after visualization by fluorescence microscopy. By analyzing microscopically identified fluorescent samples and nontransduced samples, we calculated an overall sensitivity and specificity of 90.2 and 95.0%, respectively. Real-time PCR had higher specificity and sensitivity than conventional PCR as analyzed by generalized linear models (P = 0.002 and P = 0.019, respectively). In conclusion, we found real-time PCR to have superior sensitivity and specificity compared to conventional PCR in determining transduction efficiency of hematopoietic progenitor cells.

Hematopoietic expression of O(6)-methylguanine DNA methyltransferase-P140K allows intensive treatment of human glioma xenografts with combination O(6)-benzylguanine and 1,3-bis-(2-chloroethyl)-1-nitrosourea.

The major mechanism of tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is the DNA repair protein O(6)-methylguanine DNA methyltransferase (MGMT). This repair system can be temporarily inhibited by the free base O(6)-benzylguanine (BG), which depletes cellular MGMT activity and sensitizes tumor cells and xenografts to BCNU. In clinical studies, the combination of BG and BCNU enhanced the myeloid toxicity of BCNU, thereby reducing the maximum tolerated dose. We have shown previously that retroviral expression of the P140K mutant of MGMT (MGMT-P140K) in murine and human hematopoietic cells produces significant resistance of bone marrow cells to low-dose, combination BG and BCNU treatment in vivo. In the current study, we investigated the ability of bone marrow transplantation with MGMT-P140K-transduced hematopoietic cells to protect against an intensive antitumor treatment regimen of combination BG and BCNU in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The donor marrow cells underwent in vivo BG and BCNU selection before transplantation, allowing infusion of a highly selected population of transduced cells. Tolerance to the intensive BG and BCNU treatment was markedly improved in secondary MGMT-P140K-transplanted mice (n = 19) compared to untransplanted mice (n = 15), as indicated by blood counts and survival rate. The dose-intensified BG and BCNU therapy produced significant growth delays of glioma xenografts in MGMT-P140K-transplanted mice, extending the tumor doubling time by >40 days. These results demonstrate that MGMT-P140K-transduced bone marrow protects against BG and BCNU combination therapy in vivo and allows dose-intensified treatment of tumor xenografts.

In vivo selection of human hematopoietic cells in a xenograft model using combined pharmacologic and genetic manipulations.

Strategies that increase the ability of human hematopoietic stem and progenitor cells to repair alkylator-induced DNA damage may prevent the severe hematopoietic toxicity in patients with cancer undergoing high-dose alkylator therapy. In the context of genetic diseases, this approach may allow for selection of small numbers of cells that would not otherwise have a favorable growth advantage. No studies have tested this approach in vivo using human hematopoietic stem and progenitor cells. Human CD34(+) cells were transduced with a bicistronic oncoretrovirus vector that coexpresses a mutant form of O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and the enhanced green fluorescent protein (EGFP) and transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Mice were either not treated or treated with O(6)-benzylguanine (6BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). At 8-weeks postinjection, a 2- to 8-fold increase in the percentage of human CD45(+)EGFP(+) cells in 6BG/BCNU-treated versus nontreated mice was observed in the bone marrow and was associated with increased MGMT(P140K)-repair activity. Functionally, 6BG/BCNU-treated mice demonstrated multilineage differentiation in vivo, although some skewing in the maturation of myeloid and B cells was observed in mice transplanted with granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood compared to umbilical cord blood. Expansion of human cells in 6BG/BCNU-treated mice was observed in the majority of mice previously transplanted with transduced umbilical cord blood cells. In addition, a significant increase in the number of EGFP(+) progenitor colonies in treated versus nontreated mice were observed in highly engrafted mice indicating that selection and maintenance of human progenitor cells can be accomplished by expression of MGMT(P140K) and treatment with 6BG/BCNU.

Cloning and characterization of bovine cytosolic and mitochondrial PEPCK during transition to lactation.

The cytosolic (C) and mitochondrial (M) forms of phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are encoded by two different nuclear genes in mouse, human, and chicken. Our objective was to clone the two forms of PEPCK for bovine and determine their expression during the immediate periparturient interval in dairy cows. Bovine PEPCK-C cDNA contains 2,592 nucleotides and contains 84% similarity to the coding sequence of human PEPCK-C cDNA. A 449-nt partial clone of the 3' end of PEPCK-M is 76% similar to the corresponding sequence of human PEPCK-M. The coding sequence of bovine PEPCK-C and coding sequence of the partial PEPCK-M clone were 58% similar but the similarities in the 3'-untranslated regions were negligible. Northern blot analysis revealed single transcripts of 2.85 and 2.35 kb for PEPCK-C and PEPCK-M, respectively. The transition to lactation did not alter PEPCK-M transcript expression, but expression of PEPCK-C mRNA was transiently increased during early lactation, indicating that enhanced hepatic gluconeogenesis during this period may be tied to enhanced capacity for cytosolic rather than mitochondrial formation of phosphoenolpyruvate.

Differential Secondary Reconstitution of In Vivo-Selected Human SCID-Repopulating Cells in NOD/SCID versus NOD/SCID/γ chain Mice.

Humanized bone-marrow xenograft models that can monitor the long-term impact of gene-therapy strategies will help facilitate evaluation of clinical utility. The ability of the murine bone-marrow microenvironment in NOD/SCID versus NOD/SCID/γ chain(null) mice to support long-term engraftment of MGMT(P140K)-transduced human-hematopoietic cells following alkylator-mediated in vivo selection was investigated. Mice were transplanted with MGMT(P140K)-transduced CD34(+) cells and transduced cells selected in vivo. At 4 months after transplantation, levels of human-cell engraftment, and MGMT(P140K)-transduced cells in the bone marrow of NOD/SCID versus NSG mice varied slightly in vehicle- and drug-treated mice. In secondary transplants, although equal numbers of MGMT(P140K)-transduced human cells were transplanted, engraftment was significantly higher in NOD/SCID/γ chain(null) mice compared to NOD/SCID mice at 2 months after transplantation. These data indicate that reconstitution of NOD/SCID/γ chain(null) mice with human-hematopoietic cells represents a more promising model in which to test for genotoxicity and efficacy of strategies that focus on manipulation of long-term repopulating cells of human origin.

In Vivo Measurements of Tumor Metabolism and Growth after Administration of Enzastaurin Using Small Animal FDG Positron Emission Tomography.

Background. The use of 2-[(18)F]fluoro-2-deoxy-D-glucose ([(18)F]FDG) may help to establish the antitumor activity of enzastaurin, a novel protein kinase C-beta II (PKC-betaII) inhibitor, in mouse xenografts. Methods. The hematologic cell line RAJI and the solid tumor cell line U87MG were each implanted in NOD/SCID mice. Standard tumor growth measurements and [(18)F]FDG PET imaging were performed weekly for up to three weeks after tumor implantation and growth. Results. Concomitant with caliper measurements, [(18)F]FDG PET imaging was performed to monitor glucose metabolism. Heterogeneity of glucose uptake in various areas of the tumors was observed after vehicle or enzastaurin treatment. This heterogeneity may limit the use of [(18)F]FDG PET imaging to measure enzastaurin-associated changes in xenograft tumors. Conclusion. [(18)F]FDG PET imaging technique does not correlate with standard caliper assessments in xenografts to assess the antitumor activity of enzastaurin. Future studies are needed to determine the use of [(18)F]FDG PET imaging in preclinical models.

In vivo effects of myeloablative alkylator therapy on survival and differentiation of MGMTP140K-transduced human G-CSF-mobilized peripheral blood cells.

High-intensity alkylator-based chemotherapy is required to eradicate tumors expressing high levels of O6-methylguanine DNA methyltransferase (MGMT). This treatment, however, can lead to life-threatening myelosuppression. We investigated a gene therapy strategy to protect human granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells (MPB) from a high-intensity alkylator-based regimen. We transduced MPB with an oncoretroviral vector that coexpresses MGMT(P140K) and the enhanced green fluorescent protein (EGFP) (n = 5 donors). At 4 weeks posttransplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, cohorts were not treated or were treated with low- or high-intensity alkylating chemotherapy. In the high-intensity-treated cohort, it was necessary to infuse NOD/SCID bone marrow (BM) to alleviate hematopoietic toxicity. At 8 weeks posttreatment, human CD45+ cells in the BM of mice treated with either regimen were EGFP+ and contained MGMT-specific DNA repair activity. In cohorts receiving low-intensity therapy, both primitive and mature hematopoietic cells were present in the BM. Although B-lymphoid and myeloid cells were resistant to in vivo drug treatment in cohorts that received high-intensity therapy, no human CD34+ cells or B-cell precursors were detected. These data suggest that improved strategies to optimize repair of DNA damage in primitive human hematopoietic cells are needed when using high-intensity anti-cancer therapy.