Expression of oncogenic epidermal growth factor receptor family kinases induces paclitaxel resistance and alters beta-tubulin isotype expression.

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.

Alpha 2 mRNA of Na+K+ ATPase is increased in astroctyes of rat hippocampus after treatment with kainic acid.

The Na+K+ ATPase (Na+ pump) plays a central role in regulating cation homeostasis and is thought to have an important role in cell proliferation. The multitude of subunit isoforms comprising the functional Na+K+ ATPase has raised the possibility that specific subunit isoform combinations may be involved in different cellular processes. We have investigated the involvement of the specific isoforms in neurons and glia at the site of a CNS lesion. Intracerebroventricular injection of kainic acid was used to induce neuronal cell loss and reactive gliosis in rat hippocampus and levels of Na+K+ ATPase subunit isoform mRNA levels were determined in cells of rat hippocampus using in situ hybridization. alpha 2 mRNA levels increased 35-40% in CA1 and CA3 astrocytes between 1-3 weeks after KA injection with no significant change in other subunit isoform mRNA levels. In addition alpha 3 mRNA levels in CA1 pyramidal neurons were decreased by approx. 35%. Small neurons in the CA1 and CA3 region showed no changes in mRNA levels for any of the Na+K+ ATPase subunit isoforms. These results may indicate a possible role for alpha 2 subunit isoform in the conversion of glial cells from a normal phenotype to the reactive phenotype characteristic in this model of CNS injury.

Epidermal growth factor receptor stimulation of diacylglycerol kinase.

The epidermal growth factor receptor (EGFR) activates formation of the phospholipid signal messenger phosphatidic acid (PA) on ligand binding. We explored the effects of chronic EGF stimulation on cellular PA in NIH3T3 cells expressing intact EGFR a mutant EGFR (EGFRvIII). The presence of EGFRvIII increased PA levels to twice those induced by chronic EGFR activation. Fatty acid methyl ester analysis revealed a marked increase in oleic acid containing PA. No apparent increase in phospholipase D (PLD) activity was detected, and diacylglycerol (DAG) kinase assays demonstrated a marked preference for dioleoyl DAG in the presence of activated EGFR or EGFRvIII. Levels of PA which were lower than would be predicted by DAG kinase activation are explained by increased phosphatidate phosphohydrolase activity. Specific inhibitors of EGFR kinase and DAG kinase suppressed DAG kinase activation and PA production by EGFRvIII. EGFR kinase activation by chronic exposure to ligand or by deletional mutation stimulates formation of a specific form of signalling PA.

Differential modulation of mitogen-activated protein (MAP) kinase/extracellular signal-related kinase kinase and MAP kinase activities by a mutant epidermal growth factor receptor.

A paradigm has been established whereby mutant tyrosine kinase receptors such as the v-erbB and v-fms gene products function as oncoproteins in the absence of ligand. A spontaneously occurring deletional mutant of the human epidermal growth factor receptor (EGFR-vIII) has been isolated from astrocytic neoplasms and transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is constitutively complexed with the majority of cellular GRB2, suggesting a link to the Ras-Mitogen-activated protein (MAP) kinase pathway (D. Moscatello, R. B. Montgomery, P. Sundareshan, H. McDanel, M. Y. Wong, and A. J. Wong, submitted for publication). In this report, we document that expression of EGFRvIII in fibroblasts is associated with downstream activation of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (MEK) and modest activation of p42 and p44 MAP kinases. The presence of EGFRvIII suppresses activation of p42 and p44 MAP kinases by phorbol 12-myristate 13-acetate (PMA) and serum; however, MEK activation by PMA is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase activity in EGFRvIII-transfected cells is augmented by the tyrosine phosphatase inhibitor sodium vanadate. EGFR-vIII is capable of transducing downstream signals through MAP kinase as evidenced by activation of cytoplasmic phospholipase A2 at levels similar to that induced by intact EGFR. Our results suggest that EGFR-vIII constitutively activates downstream signal transduction through MAP kinase, and this chronic stimulation of the MAP kinase pathway may represent one means by which mutant EGFR transduces an oncogenic signal.

Localization of the plasma membrane Ca(2+)-ATPase isoform PMCA3 in rat cerebellum, choroid plexus and hippocampus.

mRNA encoding rat plasma membrane Ca(2+)-ATPase isoform PMCA3 was localized in the granule cell layer of the cerebellum and in choroid plexus by in situ hybridization with an 35S-labelled oligodeoxynucleotide probe. In order to examine whether this isoform is expressed as a protein in brain, polyclonal antibodies were raised against a peptide corresponding to a C-terminal 18 amino acid sequence of PMCA3 which had been conjugated to bovine serum albumin. Using immunoblot analysis with affinity-purified antibodies, PMCA3 protein was found in rat brain microsomes and cultured neurons. The translated protein had an observed molecular mass of approximately 135 kDa, as predicted from molecular cloning studies. The pattern of localization of PMCA3 in brain using anti-peptide antibodies was consistent with findings from in situ hybridization. PMCA3-like immunoreactive sites were found in the granule cell and molecular layers of rat cerebellum and in choroid plexus, and the pattern of staining suggests that immunoreactive sites are associated with granule cell processes. This conclusion was supported by the finding that growth-associated protein-43, a protein known to be present in axons and nerve terminals, had a pattern of distribution similar to PMCA3 in the molecular layer of cerebellum. Very low levels of PMCA3-like immunoreactivity were associated with Purkinje cell soma or processes, consistent with the low levels of PMCA3 mRNA found in these neurons. PMCA3-like immunoreactivity was lower in hippocampus than in cerebellum; hippocampal CA1 region immunoreactivity was primarily associated with dendritic fields rather than with pyramidal cell bodies. The results demonstrate that a PMCA3-like protein is expressed in neurons of rat brain and is localized primarily in cell processes.

Localization of Na, K-ATPase isoforms in the hypothalamus of the rat.

In situ hybridization histochemistry with synthetic oligonucleotide probes was used to localize mRNAs encoding three isoforms of the catalytic (alpha) subunit and one isoform of the (beta) subunit of the Na, K-ATPase in rat hypothalamus using contact film radioautography. 3H-Ouabain binding to specific anatomical nuclei in the hypothalamus was determined using a quantitative radioautographic technique previously developed in our laboratory. Specific hybridization was found with oligonucleotide probes for mRNA encoding alpha 1, alpha 2, alpha 3, beta 1 isoforms of the Na, K-ATPase. High levels of hybridization signal for alpha 3 and beta 1 were found in ventromedial hypothalamus, supraoptic nucleus, paraventricular nucleus and the anterior hypothalamic area. Very low levels of hybridization for all isoforms were found in the optic chiasm. mRNAs encoding alpha 1 and alpha 2 isoforms were expressed at lower levels than alpha 3. The distribution of alpha 2 was consistent with expression in glial cells. Generally, levels of alpha 1 mRNA were higher in the arcuate nucleus than in other hypothalamic regions and very low levels were found in the anterior hypothalamic area. 3H-Ouabain binding was relatively diffuse, consistent with the localization of the synthesized Na, K-ATPase protein in cellular processes. The number of 3H-ouabain binding sites in the paraventricular nucleus was significantly lower than other hypothalamic nuclei studied. The results suggest that Na, K-ATPase isoforms may be differentially expressed in hypothalamic nuclei.

Pentoxifylline inhibits tumor necrosis factor-alpha-mediated cytotoxicity and cytostasis in L929 murine fibrosarcoma cells.

Tumor necrosis factor-alpha (TNF alpha) is recognized as a principal mediator of a variety of inflammatory conditions. In animal models, pentoxifylline attenuates the morbidity and mortality of bacterial sepsis, an effect which has been attributed to its ability to suppress the induction of TNF alpha. To determine whether pentoxifylline also directly inhibits the effects of TNF alpha, the ability to inhibit cytotoxicity on the TNF alpha-sensitive murine fibrosarcoma cell line, L929, was examined. Cell viability was assessed by crystal violet staining and cell proliferation was assessed by [3H]-thymidine uptake assay. TNF alpha induced dose-dependent cytotoxicity. At concentrations of TNF alpha of 1000 U/ml, viability at 3 days was approximately 35% of control. When L929 cells were co-incubated with TNF alpha (1000 U/ml) and pentoxifylline (1 mM), cell viability increased to approximately 75% of control (P = 0.001). At concentrations of TNF alpha of 10,000 U/ml, cell viability which was 11% of control with TNF alpha alone increased to 53% in the presence of pentoxifylline (P = 0.002). TNF alpha at 1000 and 10,000 U/ml concentrations decreased [3H]-thymidine uptake to approximately 5% of control values. Co-incubation with pentoxifylline significantly increased uptake to 13% of control at both TNF alpha concentrations (P = 0.002). Pentoxifylline did not affect the level of type I TNF alpha receptor--ligand cross-link product. However, in TNF alpha receptor binding assays, incubation with pentoxifylline 1 mM for 4 h was associated with an increase in the receptor affinity (control: KD = 0.42 nM vs pentoxifylline-treated: KD = 0.21 nM, P = 0.006), without significant change in number of type I TNF alpha receptors, suggesting that pentoxifylline affects post-receptor signalling events. We have observed that pentoxifylline prevents the TNF alpha-mediated activation of sn-2 arachidonic acid-specific cytosolic phospholipase A2, an important component of the signal transduction pathway of TNF alpha cytotoxicity. Because pentoxifylline does not inhibit all activities mediated by the type I TNF alpha receptor, its selective inhibition of post-receptor signalling may facilitate further study into the mechanisms underlying the diverse effects of TNF alpha.

Calibration of 14C-plastic standards for quantitative autoradiography with 33P.

Probes labeled with 33P have potential for widespread use in in situ hybridization because they are better able to detect relatively scarce mRNAs compared with probes labeled with 35S, but the relatively short half-life of 33P is a disadvantage when it is used as a radioactivity standard for quantitative autoradiography. To determine if plastic sections containing 14C can be used as standards for quantitative autoradiography with 33P, we co-exposed 33P-labeled liver paste sections and 14C-plastic standards to Hyperfilm beta max. The autoradiographic response of Hyperfilm beta max to these isotopes was almost identical. Second-order polynomial equations obtained from analysis of film relative optical density and radioactivity permitted derivation of tissue-equivalent radioactivity from the film optical densities produced by the 14C standards for 1-14-day exposures. These results validate the use of plastic 14C standards for quantifying 33P used in contact film autoradiography.

The plasma membrane Ca(2+)-ATPase mRNA isoform PMCA 4 is expressed at high levels in neurons of rat piriform cortex and neocortex.

Ca2+ transport mediated by the plasma membrane Ca(2+)-ATPase (PMCA) serves an important role in regulation of cytosolic-free Ca2+ in a variety of cells. Isoform PMCA4 mRNA distribution in rat brain was studied by in situ hybridization using 33P-labeled antisense oligodeoxynucleotide probes. Very high levels of hybridization were found in piriform cortex with high levels in amygdaloid nucleus and laminae 2 and 6 of cerebral cortex. Significantly lower levels were found in hypothalamic nuclei and very low or undetectable levels were found in cerebellum, habenula, olfactory bulb, thalamus, choroid plexus of the third and fourth ventricles and in CA1 and CA3 cells of the hippocampus. These results suggest that PMCA4 is not a housekeeping form of the Ca(2+)-ATPase.

Na,K-ATPase is decreased in hippocampus of kainate-lesioned rats.

The effects of intraventricular injection of kainic acid on the Na,K-ATPase (Na,K pump) were examined in discrete pyramidal cell regions of rat hippocampus. [3H]Ouabain binding was used to quantitate Na,K-ATPase catalytic subunits and in situ hybridization was used to determine Na,K-ATPase mRNA levels. Large decreases were found in both [3H]ouabain binding and alpha 3 isoform mRNA in hippocampus areas, especially in the CA3 pyramidal cell layer, which sustains heavy cell losses as a result of bilateral, intraventricular injection of kainic acid. Substantial decreases in the high affinity component of ouabain binding and in the alpha 3 isoform mRNA (but not isoforms for other Na,K-ATPase subunits) were also observed in the CA1 region of hippocampus, an area preserved in this model. High affinity [3H]ouabain binding was decreased 25-33% in the stratum pyramidale and stratum radiatum after treatment with kainic acid, and alpha 3 mRNA was decreased by 26-50%. To further characterize the decrease in alpha 3 mRNA, animals were killed at 1, 2, and 3 weeks after injection of kainate and results show a large decrease in alpha 3 mRNA only at 2 weeks recovery time. While the pathology underlying temporal lobe epilepsy is unclear, changes in the Na,K-ATPase may be involved in abnormal firing characteristics of cells in epileptic tissue.

Selection of oligonucleotide probes for detection of mRNA isoforms.

In this article we discuss strategies for selecting oligonucleotides to target isoform-specific mRNAs, drawing on our experience with isotopically labeled oligonucleotides for ISH of Na,K-ATPase mRNA alpha- and beta-subunit isoforms. Oligonucleotide probes based on one of these isoforms have a high probability of forming nonspecific hybrids with related isoform mRNAs. The design and selection of isoform-specific ISH and how their nucleotide structure influences hybridization are reviewed, as well as basic principles in identifying and evaluating candidate probes. Controls such as Tm analysis and GC content are evaluated. For distinguishing among multiple isoforms of gene families, choose lowest possible homology between isoforms consistent with other factors that influence probe performance.

Fundamentals of quantitative autoradiography by computer densitometry for in situ hybridization, with emphasis on 33P.

The goal of quantitative autoradiography (QAR) in in situ hybridization (ISH) is to determine the amount of radioactive oligonucleotide or riboprobe present in the corresponding area of the tissue slice that produced an autoradiographic image. This article discusses (a) some of the considerations related to selection and use of computer image analysis systems for accomplishing this objective, (b) development of 14C plastic autoradiographic standards for ISH QAR with 33P, (c) using QAR to develop Tm curves for ISH probes, and (d) measurement of resolution with video imaging systems for QAR.

Plasma membrane Ca(2+)-ATPase isoforms: distribution of mRNAs in rat brain by in situ hybridization.

Several mRNAs which encode for isoforms of the plasma membrane Ca(2+)-transport ATPase (PMCA) are present in adult rat brain. Using in situ hybridization with antisense oligonucleotide probes we found complex patterns of specific hybridization for three isoforms (PMCA1-3). Each rat brain region studied exhibited a distinct pattern of expression of isoforms. PMCA1 mRNA, which is widely distributed in rat tissues, was highest in CA1 pyramidal cells of hippocampus and very low in hypothalamic nuclei, cerebellum and choroid plexus. PMCA2 mRNA was highest in Purkinje cells of cerebellum and low in caudate-putamen, hypothalamic nuclei, habenula and choroid plexus. The highest levels of PMCA3 mRNA were found in habenula and choroid plexus. The PMCA1-3 isoforms appeared to be expressed primarily in neurons since hybridization was detected neither in white matter nor in regions rich in astrocytes. In different regions, different levels of expression of each PMCA mRNA may underlie specialized requirements for calcium homeostasis in specific neurons.

[3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

Localization of insulin receptor mRNA in rat brain by in situ hybridization.

Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus.

Standardization of tritium-sensitive film for quantitative autoradiography.

A method for the calibration of plastic tritium standards for use with LKB Ultrofilm is described and validated. This method uses 12-microns cryostat slices of frozen liver which have been labeled with [3H]-formaldehyde and extracted with chloroform-methanol to remove lipids. Quantitative autoradiographic measurement of 3H radioactivity in the liver slices was underestimated by 35% when lipids were not extracted. Plastic sections impregnated with tritium were calibrated in terms of lipid-extracted, tissue-equivalent radioactivity content. Calibrated standard curves for these plastic standards were closely fit (p = 0.99) by second order polynomial equations for exposures of 1, 4, 7, 13, 28, and 104 days. The equations are generally useful for any plastic tritium standards.

Localization and characterization of binding sites with high affinity for [3H]ouabain in cerebral cortex of rabbit brain using quantitative autoradiography.

[3H]Ouabain binding was studied in sections of rabbit somatosensory cortex by quantitative autoradiography and in rabbit brain microsomal membranes using a conventional filtration assay. KD values of 8-12 nM for specific high-affinity binding of [3H]ouabain were found by both methods. High-affinity binding was not uniformly distributed in somatosensory cortex and was localized predominantly to laminae 1, 3, and 4. [3H]Ouabain binding in tissue sections was stimulated by the ligands Mg2+/Pi or Mg2+/ATP/Na+ and was inhibited by K+ (IC50 = 0.7-0.9 mM), N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and erythrosin B. We conclude that [3H]ouabain is reversibly and specifically bound with high affinity in rabbit brain tissue sections under conditions that favor phosphorylation of Na+,K+-ATPase. Quantitative autoradiography is a powerful tool for assessing the affinity and number of specific ouabain binding sites in brain tissue.