Biochemical correlates of temozolomide sensitivity in pediatric solid tumor xenograft models.

The antitumor activity of the methylating agent temozolomide has been evaluated against a panel of 17 xenografts derived from pediatric solid tumors. Temozolomide was administered p.o. daily for five consecutive days at a dose level of 66 mg/kg. Courses of treatment were repeated every 21 days for three cycles. Tumor lines were classified as having high, intermediate, or low sensitivity, determined by complete responses, partial responses, or stable disease, respectively. Overall, temozolomide induced complete responses in five lines and partial responses in three additional tumor lines, giving objective regressions in 47% of xenograft lines. Analysis of temozolomide plasma systemic exposure indicated that this dose level was relevant to exposure achieved in patients. Tumors were analyzed by immunoblotting for levels of O6-methylguanine-DNA methyltransferase (MGMT) and two mismatch repair proteins, MLH-1 and MSH-2. Tumors classified as having high or intermediate sensitivity had low or undetectable MGMT and expressed detectable MLH-1 and MSH-2 proteins. Tumors classified as having low sensitivity had either (a) high MGMT or (b) low or undetectable MGMT but were deficient in MLH-1. The relationship between p53 and response to temozolomide was also examined. In vitro temozolomide did not induce p21cip1 in p53-competent NB-1643 neuroblastoma cells. Suppression of p53 function in NB1643 clones through stable expression of a trans dominant negative p53 (NB1643p53TDN) did not confer temozolomide resistance. Similarly, tumor sensitivity to temozolomide did not segregate with p53 genotype or p53 functional status. These results indicate that MGMT is the primary mechanism for temozolomide resistance, but in the absence of MGMT, proficient mismatch repair determines sensitivity to this agent.

Brain-derived neurotrophic factor promotes survival and chemoprotection of human neuroblastoma cells.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and protection against neuronal damage. We addressed whether BDNF might promote survival and chemoprotection in neuroblastoma (NB) using a drug-sensitive human NB cell line. All-trans-retinoic acid (ATRA) induces a striking phenotypic differentiation of NB1643 cells, and exogenous BDNF treatment promotes survival of these differentiated cells. ATRA induces TRKB expression, and exogenous BDNF stimulates both autophosphorylation of TRKB and induction of the immediate early gene, FOS, in these cells. BDNF mRNA is expressed in NB1643 cells. Because the time course of TRKB induction closely parallels phenotypic differentiation of these cells, it seems probable that ATRA induces differentiation of NB1643 cells by establishing an autocrine loop involving BDNF and TRKB. Exogenous BDNF treatment resulted in a further increase in neurite outgrowth, which again suggests that an autocrine loop is involved in differentiation of NB1643 cells in response to ATRA. We then tested whether BDNF might afford drug resistance in NB and found that BDNF does indeed protect in this NB model against cisplatin, a DNA-damaging agent actually used in the treatment of NB.

Brain-derived neurotrophic factor induces phosphorylation of fibroblast growth factor receptor substrate 2.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival. Gaining an understanding of how BDNF, via the tropomyosin-related kinase B (TRKB) receptor, elicits specific cellular responses is of contemporary interest. Expression of mutant TrkB in fibroblasts, where tyrosine 484 was changed to phenylalanine, abrogated Shc association with TrkB, but only attenuated and did not block BDNF-induced phosphorylation of mitogen-activated protein kinase (MAPK). This suggests there is another BDNF-induced signaling mechanism for activating MAPK, which compelled a search for other TrkB substrates. BDNF induces phosphorylation of fibroblast growth factor receptor substrate 2 (FRS2) in both fibroblasts engineered to express TrkB and human neuroblastoma (NB) cells that naturally express TrkB. Additionally, BDNF induces phosphorylation of FRS2 in primary cultures of cortical neurons, thus showing that FRS2 is a physiologically relevant substrate of TrkB. Data are presented demonstrating that BDNF induces association of FRS2 with growth factor receptor-binding protein 2 (GRB2) in cortical neurons, fibroblasts, and NB cells, which in turn could activate the RAS/MAPK pathway. This is not dependent on Shc, since BDNF does not induce association of Shc and FRS2. Finally, the experiments suggest that FRS2 and suc-associated neurotrophic factor-induced tyrosine-phosphorylated target are the same protein.

Brain derived neurotrophic factor protects human neuroblastoma cells from DNA damaging agents.

Neurotrophins are required for survival of neurons during development and may act as survival factors to cells undergoing stress. We tested whether brain derived neurotrophic factor (BDNF) protects neuroblastoma (NB) cells from cytotoxic agents using a model NB cell line, NB 1643, which expresses functional tropomyosin related kinase B (TRKB) following treatment with all-trans-retinoic acid. TRKB is the receptor for BDNF. BDNF increases the EC50 values in survival assays for cisplatin, doxorubicin, and topotecan by two to three fold. Thus, BDNF does indeed protect cells drugs that damage DNA. Cisplatin and doxorubicin are used to treat NB. Topotecan is in clinical studies for the treatment of NB. Since these drugs induce DNA damage, we also investigated whether BDNF might afford protection from gamma irradiation. BDNF also induces more than a two fold resistance to gamma irradiation. Since BDNF protects cells from agents with different mechanisms of damaging DNA and resistance, it seems likely that BDNF may alter a common signaling pathway required for cell death initiation by DNA damaging agents.

Wild-type p53 can induce p21 and apoptosis in neuroblastoma cells but the DNA damage-induced G1 checkpoint function is attenuated.

p53 is a tumor suppressor protein important in the regulation of apoptosis. Because p53 functions as a transcription factor, cellular responses depend upon activity of p53 localized in the nucleus. Cytoplasmic sequestration of p53 has been proposed as a mechanism by which the function of this protein can be suppressed, particularly in tumor types such as neuroblastoma in which the frequency of mutations of p53 is low. Data presented here demonstrate that nuclear p53 protein is expressed in a panel of neuroblastoma cell lines, and after exposure to DNA damage, transcriptionally active p53 expression can be induced. After exposure to both equitoxic IC80 and 10-Gy doses of ionizing radiation, both p53 and p21 were induced, but G1 cell cycle arrest was attenuated. To investigate whether the DNA damage signaling pathway was incapable of inducing sufficient p53 in these cells, we expressed additional wild-type p53 after adenoviral vector transduction. This exogenous p53 expression also resulted in p21 induction but was unable to enhance the G1 arrest, suggesting that the pathway downstream from p53 is nonfunctional. Although p53-mediated G1 arrest is attenuated in neuroblastoma cells, the ability of p53 to induce apoptosis appears functional, consistent with its chemosensitive phenotype. This work demonstrates that p53 is expressed in the nucleus of neuroblastoma cells and can mediate induction of p21. However, this cell type appears to have an attenuated ability to mediate a DNA damage-induced G1 cell cycle arrest.

Identification of TrkB autophosphorylation sites and evidence that phospholipase C-gamma 1 is a substrate of the TrkB receptor.

The TrkB receptor protein-tyrosine kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. In response to brain-derived neurotrophic factor and neurotrophin-3 treatment, TrkB expressed exogenously in Rat-2 cells is rapidly phosphorylated on tyrosine residues. At least 2 regions of TrkB contain phosphorylated tyrosines. The major sites of autophosphorylation are in the region containing Tyr-670, Tyr-674, and Tyr-675, which lies in the kinase domain and corresponds by sequence homology to the Tyr-416 autophosphorylation site in p60c-Src. Tyr-785, which lies just to the COOH-terminal side of the kinase domain in a relatively short tail characteristic of the Trk family of protein-tyrosine kinase receptors, is also phosphorylated in response to neurotrophin-3 treatment. The sequence around Tyr-785 fits a consensus sequence for binding phospholipase C-gamma 1. The simplest interpretation of these results is that, in response to neurotrophin binding, at least two and perhaps all three of the tyrosines in the Tyr-670/674/675 region are autophosphorylated independently, and Tyr-785 is autophosphorylated in vivo. Following activation of TrkB, phospholipase C-gamma 1 is phosphorylated on Tyr-783, Tyr-771, and Tyr-1254. Phospholipase C-gamma 1 also forms a complex with TrkB in response to neurotrophin-3 treatment, consistent with the possibility that one of the TrkB autophosphorylation sites provides a binding site for the phospholipase C-gamma 1 SH2 domains, as is the case for other receptor protein-tyrosine kinases. We conclude that phospholipase C-gamma 1 is directly phosphorylated by TrkB. Since phosphorylation of Tyr-783 and Tyr-1254 results in activation of phospholipase C-gamma 1, we predict that neurotrophin-3 leads to activation of phospholipase C-gamma 1 following binding to TrkB in Rat-2 cells.

Increased production of the TrkB protein tyrosine kinase receptor after brain insults.

The protein-tyrosine kinases Trk, TrkB, and TrkC are signal-transducing receptors for a family of neurotrophic factors known as the neurotrophins. Here we show that seizures induced by hippocampal kindling lead to a rapid, transient increase of trkB mRNA and protein in the hippocampus. TrkB is a component of a high affinity receptor for brain-derived neurotrophic factor (BDNF). No change was detected in mRNAs for Trk or TrkC, components of the high affinity nerve growth factor or neurotrophin-3 receptors, respectively. trkB mRNA was also transiently increased in the dentate gyrus following cerebral ischemia and hypoglycemic coma; these treatments had no effect on trk and trkC mRNAs. The increase in trkB mRNA and protein showed the same time course and distribution as the increase in BDNF mRNA. These data suggest that BDNF and its receptor may play a local role within the hippocampus in kindling-associated neural plasticity and in neuronal protection following epileptic, ischemic, and hypoglycemic insults.

Increased levels of trkB mRNA and trkB protein-like immunoreactivity in the injured rat and cat spinal cord.

Expression of neurotrophins and neurotrophin receptors was examined with in situ hybridization and immunohistochemical techniques 10 days to 6 weeks after ventral or dorsal funiculus spinal cord lesions in adult rats and cats, lesions that have previously been shown to allow axon regrowth. Strongly elevated levels of trkB mRNA were seen in the scar tissue formed in the white matter after both types of lesions. Only small increases were detected for nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, neurotrophin 4, trk, and trkC mRNA in response to the injuries. trkB protein-like immunoreactivity was increased in the regions that showed elevated levels of trkB mRNA. EM localized this immunoreactivity to neurons, astrocytes, and leptomeningeal cells. Neurofilament immunolabeling and axonal tracing demonstrated that nerve fibers in the scar tissue were concentrated to areas that showed strong trkB protein-like immunoreactivity. The findings implicate a role for neurotrophin receptors in axonal sprouting and glial reactions in the injured spinal cord.

Receptor protein tyrosine kinases and phosphatases.

It is clear that the number of receptor PTKs and PTPs encoded by a typical vertebrate genome is rather large. Although the signal pathways activated by the receptor PTKs may in many cases be common, specificity is provided by the ligand-binding domain and the availability of ligand. In addition, the precise spectrum of substrates that bind to and are phosphorylated by each receptor PTK can differ based on the number and nature of the autophosphorylation sites and on the repertoire of SH2-containing proteins and other substrates expressed in each cell type. It is also clear that receptor PTKs can activate multiple independent signaling pathways and that the output of these pathways can be integrated to provide a specific cellular response. The role of receptor PTPs in such integrated signaling networks is not yet obvious. In some cases, they may activate nonreceptor PTKs, whereas in other cases, they may counteract the effects of activated receptor and nonreceptor PTKs by dephosphorylating the PTKs themselves or their substrates. We know very little about the substrate specificity of PTPs, but in part this must be dictated by their subcellular location. It is possible that there are specific pairs of receptor PTKs and PTPs, which act in concert at the cell surface to activate and down-regulate specific signal pathways. Progress in understanding the function of receptor PTPs will depend on identifying ligands for receptor PTPs and then determining how ligand binding influences their activity.

The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor.

Neurotrophic factors are essential for neuronal survival and function. Recent data have demonstrated that the product of the tyrosine kinase trk proto-oncogene binds NGF and is a component of the high affinity NGF receptor. Analysis of the trkB gene product, gp145trkB, in NIH 3T3 cells indicates that this tyrosine kinase receptor is rapidly phosphorylated on tyrosine residues upon exposure to the NGF-related neurotrophic factors BDNF and NT-3. Furthermore, gp145trkB specifically binds BDNF and NT-3 in NIH 3T3 cells and in hippocampal cells, but does not bind NGF. Thus, the trk family of receptors are likely to be important signal transducers of NGF-related trophic signals in the formation and maintenance of neuronal circuits.

trkB, a neural receptor protein-tyrosine kinase: evidence for a full-length and two truncated receptors.

We have screened an adult rat cerebellar cDNA library in search of novel protein tyrosine-kinase (PTK) cDNAs. A cDNA for a putative PTK, trkB, was cloned, and its sequence indicates that it is likely to be derived from a gene for a ligand-regulated receptor closely related to the human trk oncogene. Northern (RNA) analysis showed that the trkB gene is expressed predominantly in the brain and that trkB expresses multiple mRNAs, ranging from 0.7 to 9 kb. Hybridization of cerebral mRNAs with a variety of probes indicates that there are mRNAs encoding truncated trkB receptors. Two additional types of cDNA were isolated, and their sequences are predicted to encode two distinct C-terminally truncated receptors which have the complete extracellular region and transmembrane domain, but which differ in their short cytoplasmic tails.

A major site of tyrosine phosphorylation within the SH2 domain of Fujinami sarcoma virus P130gag-fps is not required for protein-tyrosine kinase activity or transforming potential.

Phosphorylation of the major autophosphorylation site (Tyr-1073) within Fujinami sarcoma virus P130gag-fps activates both the intrinsic protein-tyrosine kinase activity and transforming potential of the protein. In this report, a second site of autophosphorylation Tyr-836 was identified. This tyrosine residue is found within a noncatalytic domain (SH2) of P130gag-fps that is required for full protein-kinase activity in both rat and chicken cells. Autophosphorylation of this tyrosine residue implies that the SH2 region lies near the active site in the catalytic domain in the native protein and thus possibly regulates its enzymatic activity. Four mutations have occurred within the SH2 domain between the c-fps and v-fps proteins. Tyr-836 is one of these changes, being a Cys in c-fps. Site-directed mutagenesis was used to investigate the function of this autophosphorylation site. Substitution of Tyr-836 with a Phe had no apparent effect on the transforming ability or protein-tyrosine kinase activity of P130gag-fps in rat-2 cells. Mutagenesis of both autophosphorylation sites (Tyr-1073 and Tyr-836) did not reveal any cooperation between these two phosphorylation sites. The implications of the changes within the SH2 region for v-fps function and activation of the c-fps oncogenic potential are discussed.

Identification of subunits of acetylcholine receptor that interact with a cholesterol photoaffinity probe.

All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with [3H]cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.

Exposure of acetylcholine receptor to the lipid bilayer.

All four subunits of the acetylcholine receptor in membrane fragments isolated from T. californica have been labeled with a photolabile hydrophobic probe, [3H]adamantanediazirine, which selectively labels regions of integral membrane proteins in contact with the hydrocarbon core of the lipid bilayer. As all of the homologous subunits are exposed to the lipid bilayer, it is probable that they each interact with the surrounding membrane in a similar fashion.