Open Access Kent State (OAKS)

Vascular endothelial growth factor (VEGF) promotes angiogenesis. VEGF has different isoforms with different functions that are generated by alternative splicing (AS).There are several proteins that participate in splicing, including serine-arginine-rich splicing factors (SRSF).SRSF are activated by kinases such as serine arginine rich protein kinases (SRPKs). Hypoxia has been shown to upregulate VEGF in several cancers. Others have demonstrated that hypoxia up-regulates SRSF proteins, which subsequently increases just the VEGF 121 isoforms in endometrial carcinoma cells. We previously showed that in hypoxic leukemia cells VEGF isoforms were spliced differently and VEGF 121 levels were inversely correlated with levels of WT1, a transcription factor. In acute lymphoblastic leukemia cells, VEGF165 and WT1 were significantly increased by hypoxia. In chronic myelogenous leukemia cells, hypoxia increased both VEGF165 and VEGF121 levels with little change in WT1 levels. Since others have shown that WT1 suppresses SRPK1 levels in sarcoma cells, our data in leukemia cells were consistent with an inverse relation between WT1 and VEGF 121 levels. The aim of this study was to determine if hypoxic conditions activated SRPK1/SRPK2 in leukemic cells to alter splicing of VEGF isoforms. The results indicated that hypoxia does not change mRNA levels of SRPK1/SRPK2, although protein levels need to be examined. Another possible explanation could be that other splicing kinases may be responsible for altering the VEGF 121 levels in leukemia cells. A better understanding of the mechanism of AS of VEGF could lead to new therapy targeting specific isoforms.

Prostate cancer (PC) is one of the most common male cancers in United States. PC lethality is due to metastatic growth, when cancer cells move from the primary tumor site to a secondary site. A key step is the acquisition of motility. Connexins (Cx) play a major role in cell-cell communication and contribute to motility. Cx43 is up-regulated in cultured PC3 metastatic prostate cancer cells. To understand the role of Cx43 in migration we asked whether silencing Connexin 43 would decrease PC3 cell migration. Using cells in which short interfering RNA (si-RNA) silenced Cx43 mRNA, we showed that silencing Cx43 decreased motility of si-Cx43 PC3 cells in Boyden chamber assays. To confirm these results in another biological system, we performed cellular wound healing assays. Silencing Cx43 decreased motility of PC3 cells reducing movement to wounded area, leaving more area open in the Cx43 inhibited cell lines. These results supported those of our Boyden assays showing that loss of Cx43 decreases the migration ability of PC3. Next we asked whether overexpression of Cx43 in LNCaP cells, non-migratory prostate cancer cells that lack Cx43 expression, would increase their motility. Currently we are working to engineer Cx43 expression in a modified LNCaP cell line. A comparison of cell lines engineered to overexpress or to silence Cx43 will confirm the importance of Cx43 in the motility of prostate cancer cells in vitro. Cx43 relevance will later be tested in vivo and could lead to new targeting strategies for men suffering from metastatic prostate cancer.

Obesity is a leading health problem in the United States, despite the abundance of weight-loss programs available. There is an increasing interest in weight-loss methods that use time-restricted feeding, including intermittent fasting, where days of limited food access are alternated with days of normal food intake. To determine the benefits of intermittent fasting to obesity, we employed a rat model of inherent leanness and obesity, with the lean rats being more physically active (high-capacity runners) than the obesity-prone (low-capacity runners). Our hypothesis stated that intermittent fasting would induce loss of fat and lean mass in both rat types with the obesity-prone rats losing more body weight. Body weight and composition were measured before intermittent fasting in lean and obesity-prone male rats (n=8/group). Rats were allowed free access to food on fed days, alternating with fasting days (no calories consumed). Rats were weighed daily and the body composition was analyzed weekly using magnetic resonance spectroscopy (EchoMRI). All rats lost weight over eight weeks of intermittent fasting. Obesity-prone rats lost significantly more fat mass and body weight compared to the lean rats, whereas loss of lean mass differed by only a few grams. Results were similar in female rats but contrasted with previous findings when weight loss was induced using 50% calorie restriction. Compared to standard calorie restriction, intermittent fasting decreased the loss of lean mass while enhancing the loss of fat mass. Intermittent fasting was thus found to be successful in inducing weight loss in obese rats.

Vascular endothelial growth factor (VEGF) is necessary for angiogenesis and tumor growth. VEGF splice isoforms have distinct characteristics, with VEGF121 being more diffusible, thus allowing for more distant angiogenic signaling, and VEGF165 and VEGF189 being found within the cell and the extracellular matrix. We hypothesized that VEGF expression in cancer cells would increase under hypoxia and that the VEGF 121 isoform would be enhanced. We examined VEGF mRNA levels and relative isoform ratios in cells grown in hypoxia or treated with CoCl2 to mimic hypoxia. Prostate cancer (PC3, LNCaP) and leukemia (MOLT-4, K562) cells were initially cultured under normoxic conditions with 20% O2 and then placed in a hypoxia chamber with 1% O2 or treated with CoCl2. RNA was extracted from treated and control cells, and quantitative real-time PCR analysis was performed to analyze VEGF expression under the different conditions. Our findings suggest that hypoxia increased both VEGF121 and VEGF165 expression in prostate cancer and leukemia cells. These results of elevated VEGF121 indicate the potential for lethal metastatic tumor growth distant from the primary tumor site. Overall, this work highlights the role of the hypoxic tumor microenvironment in regulating the functionally distinct VEGF isoforms in cancer cells.

Prostate cancer (PC) is a very common type of cancer in males and metastatic growth often leads to death. Connexins (Cx) are important to intercellular communication and may also play a role in metastasis. Cultured PC3 metastatic prostate cancer cells have high levels of Cx43 and readily migrate in Boyden chambers, a model for metastasis. We previously silenced Cx43 in PC3 cells and tested their migration in Boyden chamber assays. Silencing Cx34 reduced the motility of the migratory PC3 cells. Conversely, LNCaP prostate cancer cells are not migratory and express very little Cx43. We hypothesized that over-expressing Cx43 in LNCaP cells would enhance their motility. To test this, we transfected LNCaP cells with a Cx43 expression plasmid and used Boyden chamber migration to analyze their motility. Transfection of the LNCaP cells with the Cx43 expression plasmid increased migration 2-fold compared to the control empty expression plasmid. Knowing the role that Cx43 plays in cell motility can lead to new ways to target and treat metastatic prostate cancer.

Scientific Teaching, as described by the National Academies Scientific Teaching Alliance, is teaching science in ways that are: based on solid theory, tested and evaluated and organized in a logical framework (just like scientific research). Key elements of scientific teaching are active learning, assessment and inclusive teaching (or teaching for a diverse audience). The steps of designing a teachable unit (setting learning goals, identifying evidence for learning, planning activities and reviewing alignment) will be described with examples.