Ca++/calmodulin-dependent protein kinase II: Messenger RNA in developing rat brain

Ca$\sp{++}$/calmodulin-dependent protein kinase II (CaM-KII) is highly concentrated in mammalian brain, comprising as much as 2% of the total protein in some regions. In forebrain, CaM-KII has been shown to be enriched in postsynaptic structures where it has been implicated in maintaining cytoskeletal structure, and more recently in signal transduction mechanisms and processes underlying learning and memory. CaM-KII appears to exist as a holoenzyme composed of two related yet distinct subunits, alpha and beta. The ratio of the subunits in the holoenzyme varies with different brain regions and to some degree with subcellular fractions. The two subunits also display distinct developmental profiles. Levels of alpha subunit are not evident at birth but increase dramatically during postnatal development, while levels of beta subunit are readily detected at birth and only gradual increase postnatally. The distinct regional, subcellular and developmental distribution of the two subunits of CaM-KII have prompted us to examine factors involved in regulating the synthesis of the subunit proteins.^ This dissertation addresses the regional and developmental expression of the mRNAs for the individual subunits using in situ hybridization histochemistry and northern slot-blot analysis. By comparing the developmental profile of each mRNA with that of its respective protein, we have determined that initiation of gene transcription is likely the primary site for regulating CaM-KII protein levels. Furthermore, the distinct cytoarchitecture of the hippocampus has allowed us to demonstrate that the alpha, but not beta subunit mRNA is localized in dendrites of certain forebrain neurons. The localization of alpha subunit mRNA at postsynaptic structures, in concert with the accumulation of subunit protein, suggests that dendritic synthesis of CaM-KII alpha subunit may be important for maintaining postsynaptic structure and/or function. ^

A 3-D CT assisted Monte Carlo evaluation of intracavitary brachytherapy implants

Intracavitary brachytherapy (ICB) combined with external beam irradiation for treatment of cervical cancer is highly successful in achieving local control. The M.D. Anderson Cancer Center employs Fletcher Suit Delclos (FSD) applicators. FSD applicators contain shields to limit dose to critical structures. Dosimetric evaluation of ICB implants is limited to assessing dose at reference points. These points serve as surrogates for treatment intensity and critical structure dose. Several studies have mentioned that the ICRU38 reference points inadequately characterize the dose distribution. Also, the ovoid shields are rarely considered in dosimetry. ^ The goal of this dissertation was to ascertain the influence of the ovoid shields on patient dose distributions. Monte Carlo dosimetry (MCD) was applied to patient computed tomography(CT) scans. These data were analyzed to determine the effect of the shields on dose to standard reference points and the bladder and rectum. The hypothesis of this work is that the ICRU38 bladder and rectal points computed conventionally are not clinically acceptable surrogates for the maximum dose points as determined by MCD. ^ MCD was applied to the tandem and ovoids. The FSD ovoids and tandem were modeled in a single input file that allowed dose to be calculated for any patient. Dose difference surface histograms(DDSH) were computed for the bladder and rectum. Reference point doses were compared between shielded and unshielded ovoids, and a commercial treatment planning system. ^ The results of this work showed the tandem tip screw caused a 33% reduction in dose. The ovoid shields reduced the dose by a maximum of 48.9%. DDSHs revealed on average 5% of the bladder surface area was spared 53 cGy and 5% of the rectal surface area was spared 195 cGy. The ovoid shields on average reduced the dose by 18% for the bladder point and 25% for the rectal point. The Student's t-test revealed the ICRU38 bladder and rectal points do not predict the maximum dose for these organs. ^ It is concluded that modeling the tandem and ovoid internal structures is necessary for accurate dose calculations, the bladder shielding segments may not be necessary, and that the ICRU38 bladder point is irrelevant. ^

Role of activation of the protein tyrosine kinase, Src, in colorectal cancer chemoresistance

Advances in therapy for colorectal cancer have been hampered by development of resistance to chemotherapy. The Src family of protein tyrosine kinases has been associated with colorectal cancer development and progression. Activation of the prototypic member of the family, Src, occurs in advanced colorectal cancer and is associated with a worse outcome. This work tests the hypotheses that Src activation contributes to chemoresistance in some colon tumors and that this resistance can be overcome by use of Src inhibitors. The aims of the proposal were to (1) determine if constitutive Src activation is sufficient to induce oxaliplatin resistance; (2) evaluate the role of reactive oxygen species (ROS) in the activation of Src after oxaliplatin treatment; (3) determine the frequency of Src activation in liver metastases after oxaliplatin treatment; and (4) evaluate the safety, preliminary efficacy, and pharmacodynamics of the combination of dasatinib with oxaliplatin-based therapy in patients with metastatic colorectal cancer. ^ Using a panel of colon cancer cell lines and murine models, I demonstrate that administration of oxaliplatin, a commonly utilized chemotherapy for colorectal cancer, results in an increased activation of Src. The activation occurs acutely in some, but not all, colorectal carcinoma cell lines. Cell lines selected for oxaliplatin resistance are further increased in Src activity. Treatment of cell lines with dasatinib, a non-selective pharmacologic inhibitor of the Src family kinases synergistically killed some, but not all cell lines. Cell lines with the highest acute activation of Src after oxaliplatin administration were the most sensitive to the combination therapy. Previous work demonstrated that siRNA to Src increased sensitivity to oxaliplatin, suggesting that the effects of dasatinib are primarily due to its ability to inhibit Src in these cell lines. ^ To examine the mechanism underlying these results, I examined the effects of reactive oxygen species (ROS), as previous studies have demonstrated that platinum chemotherapeutics result in intracellular oxidative stress. I demonstrated that oxaliplatin-induced reactive oxygen species were higher in the cell lines with Src activation, relative to those in which Src was not activated. This oxaliplatin-induced Src activation was blocked by the administration of anti-oxidants, thereby demonstrating that synergistic killing between dasatinib and oxaliplatin was associated with the ability of the latter to generate ROS. ^ In a murine model of colorectal cancer metastasis to the liver, the combination of dasatinib and oxaliplatin was more effective in reducing tumor volume than either agent alone. However, when oxaliplatin resistant cell lines were treated with a combination of oxaliplatin and AZD0530, an inhibitor in the clinic with increased specificity for Src, no additional benefit was seen, although Src was activated by oxaliplatin and Src substrates were inhibited. The indolent growth of oxaliplatin-resistant cells, unlike the growth of oxaliplatin resistant tumors in patients, precludes definitive interpretation of these results. ^ To further explore Src activation in patients with oxaliplatin exposure and resistance, an immunohistochemistry analysis of tumor tissue from resected liver metastases of colorectal cancer was performed. Utilizing a tissue microarray, staining for phosphorylated Src and FAK demonstrated strong staining of tumor relative to stromal and normal liver. In patients recently exposed to oxaliplatin, there was increased FAK activation, supporting the clinical relevance of the prior preclinical studies. ^ To pursue the potential clinical benefit of the combination of Src inhibition with oxaliplatin, a phase IB clinical trial was completed. Thirty patients with refractory metastatic colorectal cancer were treated with a combination of 5-FU, oxaliplatin, an epidermal-growth factor receptor monoclonal antibody, and dasatinib. The recommended phase II dose of dasatinib was established, and toxicities were quantified. Pharmacodynamic studies demonstrated increased phosphorylation of the Src substrate paxillin after dasatinib therapy. Tumor biopsies were obtained and Src expression levels were quantitated. Clinical benefit was seen with the combination, including a response rate of 20% and disease control rate of 56%, prompting a larger clinical study. ^ In summary, although Src is constitutively activated in metastatic colorectal cancer, administration of oxaliplatin chemotherapy can further increase its activity, through a reactive oxygen species dependent manner. Inhibition of Src in combination with oxaliplatin provides additional benefit in vitro, in preclinical animal models, and in the clinic. Further study of Src inhibition in the clinic and identification of predictive biomarkers of response will be required to further advance this promising therapeutic target. ^

Nitrogen dioxide exposures among a selected group of residents of substandard housing in Houston, Texas

Nitrogen dioxide (NO$\sb2)$ levels in sixteen substandard houses located in Houston, Texas were examined. The classification of the houses as substandard was based on an assessment of structural integrity which would affect air exchange rates. In these homes, unvented gas space heaters were operated as the primary source of heat.^ The Ogawa passive sampling device was used to measure NO$\sb2$ concentrations over 24 to 48-hour periods during generally cold weather. A sampler was placed in the kitchen and bedroom of each house. The female head of household was asked to wear a monitor during area monitoring to assess her personal exposure. Outdoor levels of NO$\sb2$ were also measured.^ Mean (standard deviation) levels of kitchen, bedroom and personal exposures were 280 (125) ppb, 256 (155) ppb and 164 (102) ppb, respectively. Additional short-term ($<$24 hours) samples were measured in three houses. The mean level of NO$\sb2$ measured outdoors was 51 ppb over the course of the study.^ The measurements obtained with the Ogawa sampler were compared to those levels obtained using a reference method (chemiluminescence). Outdoor levels measured with the diffusion samplers were 48% higher.^ These results suggest that wintertime NO$\sb2$ levels within substandard houses using gas appliances for heating and cooking are extremely elevated. Further work is needed to investigate the prevalence of possible health effects associated with these exposures. ^

The cDNA cloning of murine HIP /L29 and the functional analysis of HIP/L29 domains

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is a heparin/heparan sulfate (Hp/HS) binding protein found in many adult human tissues. Potential functions of this protein are promotion of embryo adhesion, modulation of blood coagulation, and control of cell growth. While these activities are diverse, the ability of human HIP/L29 to interact with Hp/HS at the cell surface may be a unifying mechanism of action since Hp/HS influences all of these processes. A murine ortholog has been identified that has 78.8% homology over the entire sequence and identity over the N-terminal 64 amino acids when compared to human HIP/L29. Northern, Western, and immunohistochemical analysis shows that murine HIP/L29 mRNA and protein are expressed in a tissue specific manner. Murine HIP/L29 is enriched in the membrane fraction of NmuMG cells where it is eluted with high salt, suggesting that it is a peripheral membrane protein. The ability of murine HIP/L29 to bind Hp is verified by studies using native and recombinant forms of murine HIP/L29. A synthetic peptide (HIP peptide-2) derived from the identical N-terminal region of HIP/L29 proteins was tested for the ability to bind Hp and support cell adhesion. This peptide was chosen because it conforms to a proposed consensus sequence for Hp/HS binding peptides. HIP peptide-2 binds Hp in a dose-dependent, saturable, and selective manner and supports Hp-dependent cell adhesion. However, a scrambled form of this peptide displayed similar activities indicating a lack of peptide sequence specificity required for activity. Lastly, an unbiased approach was used to identify sequences within human and mouse HIP/L29 proteins necessary for Hp/HS binding. A panel of recombinant proteins was made that collectively are deficient in every human HIP/L29 domain. The activities of these deletion mutants and recombinant murine HIP/L29 were compared to the activity of recombinant human HIP/L29 in a number of assays designed to look at differences in the ability to bind Hp/HS. These studies suggest that each domain within human HIP/L29 is important for binding to Hp/HS and divergences in the C-terminus of human and mouse HIP/L29 account for a decrease in murine HIP/L29 affinity for Hp/HS. It is apparent that multiple domains within human and mouse HIP/L29 contribute to the function of Hp/HS binding. The interaction of multiple HIP/L29 domains with Hp/HS will influence the biological activity of HIP/L29 proteins. ^