A retrospective review of an epidural blood patch database: the incidence of epidural blood patch associated with obstetric neuraxial anesthetic techniques and the effect of blood volume on efficacy.

BACKGROUND: The optimal volume of blood required to treat post-dural puncture headache remains in question. In our institution a target volume of 30mL is used for an epidural blood patch unless the patient experiences pain during injection. METHODS: The institutional database was retrospectively reviewed for epidural blood patch and delivery statistics over a 15-year period to determine if the volume of blood administered during the procedure directly correlated with the number of epidural blood patches administered. The primary endpoint was defined as the need for a repeat epidural blood patch. RESULTS: There were 466 epidural blood patches performed on 394 patients, associated with 84 804 obstetric neuraxial procedures. Thirty-two percent (95% CI 28.3 to 34.9%) of patients who had an inadvertent dural puncture with an epidural needle received an epidural blood patch versus 0.19% (0.16% to 0.22%) of patients who received neuraxial anesthesia with no documented dural puncture with an epidural needle. All patients experienced relief of post-dural puncture headache, although 17% required two and 1.5% required three epidural blood patches. The mean±SD volume of blood administered was 20.5±5.4mL and only 35 patients (8.9%) received 30mL. CONCLUSION: Increasing blood volumes up to 30mL did not reduce the need for repeat epidural blood patch. Although the optimal volume of blood to administer during epidural blood patch placement remains unknown, our institution will continue to administer up to 30mL or until the patient experiences pain during epidural injection.

Ceramide-induced G2 arrest in rhabdomyosarcoma (RMS) cells requires p21Cip1/Waf1 induction and is prevented by MDM2 overexpression.

The sphingoplipid ceramide is responsible for a diverse range of biochemical and cellular responses including a putative role in modulating cell cycle progression. Herein, we describe that an accumulation of ceramide, achieved through the exogenous application of C(6)-ceramide or exposure to sphingomyelinase, induces a G(2) arrest in Rhabdomyosarcoma (RMS) cell lines. Utilizing the RMS cell line RD, we show that this G(2) arrest required the rapid induction of p21(Cip1/Waf1) independent of DNA damage. This was followed at later time points (48 h) by the commitment to apoptosis. Apoptosis was prevented by Bcl-2 overexpression, but permitted the maintenance of elevated p21(Cip1/Waf1) protein expression and the stabilization of the G(2) arrest response. Inhibition of p21(Cip1/Waf1) protein synthesis with cyclohexamide (CHX) or silencing of p21(Cip1/Waf1) with siRNA, prevented ceramide-mediated G(2) arrest and the late induction of apoptosis. Further, adopting the recent discovery that murine double minute 2 (MDM2) controls p21(Cip1/Waf1) expression by presenting this CDK inhibitor to the proteasome for degradation, RD cells overexpressing MDM2 abrogated ceramide-mediated p21(Cip1/Waf1) induction, G(2) arrest and the late ensuing apoptosis. Collectively, these data further support the notion that ceramide accumulation can modulate cell cycle progression. Additionally, these observations highlight MDM2 expression and proteasomal activity as key determinants of the cellular response to ceramide accumulation.

Stem cell-associated genes are extremely poor prognostic factors for soft-tissue sarcoma patients.

Cancer stem cells can play an important role in tumorigenesis and tumor progression. However, it is still difficult to detect and isolate cancer stem cells. An alternative approach is to analyse stem cell-associated gene expression. We investigated the coexpression of three stem cell-associated genes, Hiwi, hTERT and survivin, by quantitative real-time-PCR in 104 primary soft-tissue sarcomas (STS). Multivariate Cox's proportional hazards regression analyses allowed correlating gene expression with overall survival for STS patients. Coexpression of all three stem cell-associated genes resulted in a significantly increased risk of tumor-related death. Importantly, tumors of patients with the poorest prognosis were of all four tumor stages, suggesting that their risk is based upon coexpression of stem cell-associated genes rather than on tumor stage.

Ocular pharmacology of bicyclic hexahydroaporphines.

This paper explores the ocular hypotensive actions of bicyclic analogs of hexahydroaporphine (HHA), specifically nor-HHA, in an attempt to shed light on the mechanism(s) by which they lower intraocular pressure (IOP). Studies involving the measurement of IOP and aqueous humor production were conducted in ocular normotensive albino rabbits, while those involving smooth muscle contractility utilized isolated bovine iris. The ability of nor-HHA to produce a sustained drop in IOP is linked to both a functioning adrenergic nervous system and the availability of the products of cyclooxygenase metabolism. Although aqueous flow is not impacted by the bicyclic structures, the significant enhancement of outflow facility points to a probable mechanism of IOP-lowering action. Nor-HHA had no direct contractile or relaxant action on bovine irides, but does cause a concentration-dependent inhibition of carbachol-evoked contractions. This inhibition was reversed by inhibitors of phospholipase A(2) and cyclooxygenase, but not by inhibitors of lipoxygenase, again indicating a role for prostaglandins in the ocular pharmacological action of bicyclic HHAs. Pretreatment with a nitric oxide (NO) scavenger also reversed the ability of nor-HHA to inhibit carbachol-induced contractions, implying a role for NO in the postjunctional actions of HHAs.

MDM2 splice variants and their therapeutic implications.

MDM2 splice variants have now been identified in many different tumor types, and their expression has been associated with advanced disease. However, published data concerning their function is contradictory, and therefore their role in tumorigenesis and their potential as a therapeutic target are unclear. Expression of a specific splice variant, MDM2-B, in a transgenic mouse model results in tumor development; and expression of several splice variants has been shown to enhance tumor formation in Emu-myc transgenic mice. However, expression of similar variants in vitro results in growth inhibition, an observation inconsistent with a transformed phenotype. The observed growth inhibition is p53-dependent, resulting from the binding of splice variants with an intact C-terminal RING finger domain to full-length MDM2 protein. In doing so, p53 can no longer bind MDM2, and p53 activity is elevated. Subsequent inactivation of p53 or p53-mediated apoptosis could contribute to the MDM2 splice variant-mediated tumorigenesis observed in vivo. However, MDM2 splice variants, like full-length MDM2, probably display p53-independent activities. Therefore, the potential for MDM2 splice variants as therapeutic targets will be dependent upon their phenotype within specific tumor types.

Potentiation of sympathetic neurotransmission in bovine isolated irides by isoprostanes.

Isoprostanes (IsoP) are formed by free radical catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. In the present study, we examined the effect of IsoP on norepinephrine (NE) release from the bovine isolated iris. Furthermore, we studied the role of IsoP's in hydrogen peroxide (H2O2)-induced enhancement of NE release from this tissue. Isolated bovine irides were prepared for studies of [3H]NE release using the superfusion method. Release of [3H]NE was induced via electrical field stimulation. Both 8-iso-prostaglandin E2 (E2-IsoP) and 8-iso-prostaglandin F2 alpha (F2-IsoP) produced a concentration-related enhancement of field-stimulated [3H]NE release from isolated bovine irides, an effect that was mimicked by the thromboxane (Tx) receptor agonist, U46619 and by H2O2. The Tx-receptor antagonist, SQ 29548 inhibited responses to E2-IsoP (10 microM) with an IC50 of 370 +/- 50 nM. SQ 29548 (10 microM) also blocked the enhancement of electrically-evoked [3H]NE release induced by U46619 (10 microM) but not that caused by H2O2 (300 microM). The Tx synthetase inhibitor, carboxyheptylimidazole (10 microM) prevented the stimulatory effect of E2-IsoP on evoked [3H]NE release without affecting responses induced by H2O2. We conclude that IsoP's can enhance sympathetic neurotransmission in the bovine isolated iris, an effect that can be blocked by a Tx-receptor antagonist. Furthermore, endogenously produced Tx's mediate the stimulatory effect of IsoP's on NE release. However, endogenously generated IsoP's or Tx's are not involved in H2O2-induced potentiation of sympathetic neurotransmission.

Sensitization of human tumor cells to CPT-11 via adenoviral-mediated delivery of a rabbit liver carboxylesterase.

Irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) is activated by carboxylesterases (CE) to yield the potent topoisomerase I inhibitor, SN-38. We have demonstrated previously that a rabbit liver CE is approximately 100-1000-fold more efficient at drug activation than a highly homologous human CE. In an attempt to use rabbit CE expression in combination with CPT-11 for gene therapy approaches for the treatment of cancer, we have developed an adenoviral vector expressing this intracellular CE. After transduction, this virus produces very high levels of CE activity in a panel of human tumor cell lines and results in marked sensitization to CPT-11 of all of the transduced cells. Reductions in IC(50) values for this drug ranged from 11-127-fold. Additionally, comparison with an adenovirus expressing a secreted form of the rabbit CE indicated that a collateral effect could be achieved with reductions in the IC(50) values ranging from 4-19-fold. These data suggest that the described reagents may be suitable for use in vivo in a viral-directed enzyme prodrug therapy approach using CPT-11.

A virus-directed enzyme prodrug therapy approach to purging neuroblastoma cells from hematopoietic cells using adenovirus encoding rabbit carboxylesterase and CPT-11.

Tumor cells that contaminate hematopoietic cell preparations contribute to the relapse of neuroblastoma patients who receive autologous stem cell rescue as a component of therapy. Therefore, effective purging methods are needed. This study details in vitro experiments to develop a viral-directed enzyme prodrug purging method that specifically targets neuroblastoma cells. The approach uses an adenovirus to deliver the cDNA encoding a rabbit liver carboxylesterase that efficiently activates the prodrug irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11). The data show that an adenoviral multiplicity of infection of 50 transduces 100% of cultured neuroblastoma cells and primary tumor cells, irrespective of the level of tumor cell line contamination. Exposure of neuroblastoma cell lines or of mixtures of these cell lines with CD34(+) cells at a ratio of 10:90 to replication-deficient AdRSVrCE for 24 h and subsequent exposure of cells to 1-5 microM CPT-11 for 4 h increased the toxicity of CPT-11 to three neuroblastoma cell lines (SJNB-1, NB-1691, and SK-N-SH) from approximately 20-50-fold and eradicated their clonogenic potential. Also, after "purging," RNA for neuroblastoma cell markers (tyrosine hydroxylase, synaptophysin, and N-MYC) was undetectable by reverse transcription-PCR. In contrast, the purging protocol did not affect the number or type of colonies formed by CD34(+) cells in an in vitro progenitor cell assay. No bystander effect on CD34(+) cells was observed. The method described is being investigated for its potential clinical utility, particularly its efficacy for use with patients having relatively high tumor burdens, because no published methods have been shown to be efficacious when the tumor burden exceeds 1%.

[(3)H]-serotonin release from bovine iris-ciliary body: pharmacology of prejunctional serotonin (5-HT(7)) autoreceptors.

In the present study, we investigated the pharmacological characteristics of electrically stimulated [(3)H]-serotonin release from mammalian iris-ciliary bodies. Isolated bovine and human iris-ciliary bodies were loaded with [(3)H]-serotonin, superfused with Krebs buffer solution and then stimulated with trains of 300 direct current (d.c.) pulses to initiate the release of the transmitter. The modification of this [(3)H]-serotonin release process by various serotonergic agonists and antagonists was studied in order to define the pharmacology of serotonin receptor(s) present in the iris-ciliary body. In bovine iris-ciliary body, electrically-evoked [(3)H]-serotonin release was calcium-dependent, tetrodotoxin-sensitive and was enhanced by serotonin (EC(50) = 200 n M) and 5-carboxmidotryptamine (EC(50) = 4 n M). The rank order of potency of agonists in enhancing field-stimulated [(3)H]-serotonin release was: 5-carboamidotryptamine > m-chlorophenylbiguanide > 2-methyl-5-hydroxytryptamine = 5-methoxy-dimethyltryptamine > serotonin > 5-methoxy-tryptamine > L-694,247 = alpha-methyl-5-hydroxytryptamine > CGS 12066A = 8-hydroxy-2-(di- n -propylamino)tetraline. Serotonin and m-chlorophenylbiguanide also enhanced electrically-evoked [(3)H]-serotonin release from human iris-ciliary bodies with EC(50)s of 3 microM and 30 n M, respectively. The pharmacological profile displayed by serotonin receptor agonists was supported by the potent antagonism of the serotonin-induced enhancement of [(3)H]-serotonin release by 5HT(7)receptor antagonists SB-258718 (IC(50) = 18.6 +/- 1.2 nM; n = 4) and mesulergine (IC(50) = 0.26 +/- 0.05 nM; n = 4). However, antagonists at 5HT(6)and 5HT(3)receptors exhibited a relatively weak blockade of serotonin induced enhancement of field-stimulated [(3)H]-serotonin release. These studies have shown the presence of functionally active prejunctional 5HT(7)autoreceptors regulating the release of [(3)H]-serotonin from bovine iris-ciliary bodies. Excitatory prejunctional 5-HT autoreceptors also exist in human iris-ciliary bodies. It is possible that these serotonin autoreceptors may have relevance to the regulation of aqueous humor dynamics in the anterior uvea.

Use of a modified ornithine decarboxylase promoter to achieve efficient c-MYC- or N-MYC-regulated protein expression.

One of the advantages of viral-directed enzyme prodrug therapy (VDEPT) is its potential for tumor-specific cytotoxicity. However, the viruses used to deliver cDNAs encoding prodrug-activating enzymes transduce normal cells as well as tumor cells, and several approaches to achieve tumor-specific expression of the delivered cDNAs are being investigated. One such approach is to regulate transcription of the prodrug-activating enzyme with a promoter that is preferentially activated by tumor cells. Published data suggest that the most promising transcription factor/promoter/enhancer combinations are those activated by a tumor-specific transcription factor to retain tumor cell specificity but that are equal in strength to nonspecific viral promoters in their ability to up-regulate target cDNAs. This report identifies MYC-responsive, modified ornithine decarboxylase (ODC) promoter/enhancer sequences that up-regulate target protein expression in tumor cells overexpressing either N-MYC or c-MYC protein. The most efficient of the four constructs assessed contained six additional CACGTG MYC binding sites 5' to the endogenous ODC promoter (R6ODC). Reporter assays with this chimeric promoter/enhancer regulating expression of chloramphenicol acetyltransferase demonstrated 50-250-fold more activity in MYC-expressing cells compared with similar assays with promoterless plasmids. The R6ODC regulatory sequence was approximately equivalent to the CMV promoter in inducing expression of the neomycin resistance gene in c-MYC-expressing SW480 and HT-29 colon carcinoma cells and in N-MYC-expressing NB-1691 neuroblastoma cells. The modified ODC promoter may, therefore, be useful in achieving tissue-specific expression of target proteins in tumor cells that overexpress c- or N-MYC.

Hypoxia-induced [(3)H]D-aspartate release from isolated bovine retina: modulation by calcium-channel blockers and glutamatergic agonists and antagonists.

PURPOSE: The aim of the present study was two-fold: (a) to examine the effect of hypoxia on [(3)H]D-aspartate release from isolated bovine and human retinae, and (b) to investigate the regulation of hypoxia-induced neurotransmitter release by glutamate receptor agonists and antagonists. METHODS: Isolated neural retinae were incubated in oxygenated Krebs buffer solution containing [(3)H]D-aspartate and then prepared for studies of neurotransmitter release using the superfusion method. Release of [(3)H]D-aspartate was evoked by K(+) (50 mM) applied at 90 minutes (S(1)) and hypoxia (induced by exposure of tissues to solutions pregassed with 95%N(2): 5% CO(2) for 60 minutes) at 108 minutes (S(2)) after onset of superfusion. RESULTS: Under hypoxic conditions, pO(2) in normal Krebs buffer solution was reduced from 14.53 +/- 0.26 ppm (n = 6) to 0.54 +/- 0.04 ppm (n = 9) after one hour of gassing with 95% N(2): 5% CO( 2). Exposure to hypoxia elicited an overflow of [(3)H]D-aspartate yielding S(2)/S(1) ratios of 0.62 +/- 0.06 (n = 12) and 0.54 +/- 0.03 (n = 8) in bovine and human tissues respectively. In isolated bovine retinae, L- and N-calcium-channel antagonists diltiazem, nitrendipine, verapamil and omega-conotoxin significantly (p < 0.01 or higher) attenuated hypoxia-induced [(3)H]D-aspartate release. L-glutamate (30 microM) significantly (p < 0.001) potentiated hypoxia-induced [(3)H]D-aspartate release whereas kainate (30 microM) inhibited this response. NMDA (in concentrations up to 1 mM) had no effect on hypoxia-induced [(3)H]D-aspartate release. Antagonists of glutamate receptors and the polyamine site on the NMDA receptor inhibited hypoxia-induced release of [(3)H]D-aspartate in bovine retina with the following rank order of activity: ifenprodil congruent with MCPG > L-AP3 > MK-801. At an equimolar concentration (10 microM), L-AP3 but not ifenprodil, MCPG, MK 801 or arcaine, caused a significant (p < 0.001) inhibition of hypoxia-induced [(3)H]D-aspartate release from human retinae. CONCLUSIONS: Hypoxia can induce the release of [( 3)H]D-aspartate from isolated bovine retinae by a calcium-dependent process. Hypoxia-induced [(3)H]D-aspartate release from isolated bovine retinae can be regulated by glutamate receptor agonists/antagonists and blockers of polyamine site on the NMDA receptor.

Novel mdm2 splice variants identified in pediatric rhabdomyosarcoma tumors and cell lines.

Mdm2 is an oncogene that binds to and inactivates the tumor suppressor p53. However, the presence of oncogenic splice variants of mdm2 in human tumors that lack the p53 binding site has suggested a p53-independent transforming function for this protein. This report describes expression of 11 different mdm2 splice variants in pediatric rhabdomyosarcoma (RMS) cell lines and tumors at a frequency of 75% and 82%, respectively. Five of these isoforms have previously been described in other tumor histiotypes but six are novel and may be unique to RMS. There was no association between expression of splice variants and mdm2 gene amplification or p53 status. In addition, the frequency of splice variants was much higher than the incidence of mdm2 amplification or p53 mutations. These variants may be important to consider with respect to RMS tumor progression and therapeutic response.

Construction of adenovirus for high level expression of small RNAs in mammalian cells. Application to a Bcl-2 ribozyme.

A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA sequences. A truncated human U6 gene containing convenient restriction sites has been shown to be expressed at high levels following electroporation into a series of human cell lines. This gene was ligated into a promoterless adenoviral plasmid, and we have generated high titer virus by homologous recombination with adenoviral Addl327 DNA in 293 cells. Recombinant adenovirus containing a hammerhead ribozyme sequence targeted toward the Bcl-2 mRNA has been used to transduce a panel of human tumor cell lines. We have demonstrated high level expression of the recombinant U6 gene containing the ribozyme and reduction of Bcl-2 protein in transduced cells. These plasmids are suitable for the development of adenoviral vectors designed to express both ribozymes and antisense RNA in human cells.

P53 mutation and MDM2 amplification frequency in pediatric rhabdomyosarcoma tumors and cell lines.

BACKGROUND: The p53 tumor suppressor gene is the most commonly mutated gene in human cancer, and mutations arise in a wide variety of tumor types. Wild-type p53 functions as a regulator of apoptosis, so mutations in the p53 gene are generally associated with aggressive tumors and a poor prognosis. PROCEDURE: We have investigated the p53 mutation and MDM2 amplification frequencies in biopsies from pediatric rhabdomyosarcoma (RMS) tumors and cell lines by SSCP and Southern analyses. RESULTS: A mutation was detected in only 1 of 20 tumor specimens (5%), whereas the frequency in established RMS cell lines was significantly higher (6/10, 60%). p53 Mutations were more common in cell lines derived from tumors previously exposed to chemotherapy compared to those derived from tumors at di-agnosis, and it is likely that these mutations enhanced the probability of successful long-term culture. The frequency of MDM2 gene amplification in patient biopsies was also low (2/20, 10%). Interestingly, complete responses to treatment were obtained in the two patients with tumors that demonstrated amplification of MDM2. The response to treatment of patients with tumors wild-type for p53 and without MDM2 amplification was quite varied, indicating that expression of a wild-type p53 gene at diagnosis cannot always facilitate a favorable outcome. CONCLUSIONS: p53 mutation and MDM2 gene amplification frequencies are extremely low in RMS tumors, but a wild-type p53 genotype is not always associated with a favorable prognosis.

Use of the ornithine decarboxylase promoter to achieve N-MYC-mediated overexpression of a rabbit carboxylesterase to sensitize neuroblastoma cells to CPT-11.

Overexpression of specific transcription factors by tumor cells can be exploited to regulate expression of proteins that induce apoptosis or activate prodrugs, thereby producing tumor-selective toxicity. A majority of advanced-stage neuroblastomas overexpress the transcription factor N-MYC, and this overexpression is associated with poor prognosis. This study describes regulation of expression by N-MYC, via the ornithine decarboxylase (ODC) promoter, of a rabbit liver carboxylesterase (CE) that activates the prodrug CPT-11. Chloramphenicol acetyltransferase reporter assays and CE activity assays in transiently transfected neuroblastoma cell lines (SJNB-1, SJNB-4, NB-1691) and rhabdomyosarcoma cell lines (JR1neo20, JR1Nmyc6, JR1Nmyc9) support this approach as a potential method for sensitizing tumor cells to CPT-11. Clonogenic assays with IMR32 human neuroblastoma cells which express N-MYC and that had been stably transfected with a plasmid containing an ODC promoter/CE cassette corroborated results of enzyme activity assays. Specifically, IMR32.ODC.CE cells expressed approximately eightfold more CE activity than IMR32.CMV.neo cells; and 5 microM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 microM CPT-11 was required to produce the same effect with IMR32.CMV.neo cells. Current experiments focus on adenoviral delivery of an ODC promoter/CE cDNA cassette for potential virus-directed enzyme prodrug therapy applications.

Effect of isoprostanes on sympathetic neurotransmission in the human isolated iris-ciliary body.

Isoprostanes (IsoP's) are prostaglandin-like compounds that are derived from free-radical catalyzed peroxidation of arachidonic acid independent of the cyclcooxygenase enzyme. In the present study, we investigated the effect of IsoP's on norepinephrine (NE) release from human isolated iris-ciliary bodies. Isolated human iris-ciliary bodies were prepared for studies of [3H]NE release using the superfusion method. Both 8-iso-prostaglandin F2alpha (F2-IsoP) and the thromboxane (Tx) receptor agonist, U46619 enhanced field-stimulated [3H]NE release from isolated, superfused human iris-ciliary bodies without affecting basal tritium efflux. On the other hand, an equimolar concentration (10 microM) of 8-iso-prostaglandin E2 (E2-IsoP) inhibited evoked [3H]NE overflow. The Tx-receptor antagonist, SQ 29548 blocked the enhancements of electrically-evoked [3H]NE release induced by F2-IsoP and U46619. However, the inhibitory responses elicited by E2-IsoP was not antagonized by SQ 29548. We conclude that IsoP's can produce both excitatory and inhibitory effects on sympathetic neurotransmission in human isolated iris-ciliary bodies. The stimulatory effects of IsoP's on NE release may be mediated by Tx-receptors.

Effect of inhibition of cyclooxygenase on pre- and postjunctional actions of peroxides in the iris-ciliary body.

In the present study, we investigated the effect of inhibition of cyclooxygenase (COX) with flurbiprofen (FBF) on peroxide-induced enhancement of field-stimulated [3H]norepinephrine ([3H]NE) release from bovine isolated irides. Furthermore, the effect of FBF was examined on peroxide-induced attenuation of contractions evoked by carbachol on this tissue. Irides were prepared for studies of neurotransmitter release and for measurement of contractile tension in vitro. Pretreatment of tissues with FBF (10 microM) caused significant (P < 0.001) rightward shifts of concentration-response curves to H2O2 and also decreased cumene hydroperoxide (cuOOH)-induced enhancement of evoked [3H]NE release. FBF (10 microM) partially prevented the attenuation of carbachol-induced contractions induced by H2O2 (300 microM) and cuOOH (300 microM). We conclude that inhibition of the biosynthesis of prostanoids reduced both the prejunctional stimulatory effects of H2O2 and cuOOH on sympathetic neurotransmission and inhibitory effects of peroxides on carbachol-induced contractions the in the bovine isolated iris.

Wild-type p53 induction mediated by replication-deficient adenoviral vectors.

Replication-deficient E1-/E3-deleted adenoviral vectors are commonly used to introduce transgenes into cells in vitro and have been used for certain kinds of gene therapy protocols in vivo. We have demonstrated that transduction of cells using these vectors can induce p53 expression in cells containing a wild-type p53 gene. This response is different from p53 induction observed after DNA damage because the time course of induction is slower and because it occurs in cells with an attenuated DNA damage response. However, this vector-induced p53 is transcriptionally active and, therefore, p53 function is not inactivated by viral proteins. The mechanism of induction appears to be an increased rate of protein translation because immunoprecipitation analyses demonstrated increased levels of 35S-labeled p53 protein, even after a short 15-min labeling time. Induction of p53 by adenoviral vectors may have various effects on transduced cells, including apoptosis and altered chemotherapy chemosensitivity. Therefore, the influence of the vector might confound the impact of any particular gene used in a gene therapy application.

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.

Bax is an important determinant of chemosensitivity in pediatric tumor cell lines independent of Bcl-2 expression and p53 status.

Susceptibility of a tumor cell to undergo chemotherapy-induced apoptosis appears to be dependent upon the balance of proapoptotic and survival factors that are expressed within any given cell. We have chosen to evaluate how expression of several of these proteins influences chemosensitivity of a panel of 10 pediatric tumor cell lines chosen from three tumor histiotypes: neuroblastoma, rhabdomyosarcoma, and pediatric glial tumors. The proteins evaluated were p53 and six members of the Bax/Bcl-2 family: three proapoptotic proteins (Bax, Bak, and Bcl-xS) and three survival factors (Bcl-2, Bcl-xL, and Mcl-1). We investigated whether there was any relationship between endogenous expression of these proteins and chemosensitivity (or resistance) to three chemotherapeutic agents that directly damage DNA (doxorubicin, actinomycin D, and topotecan) and a mitotic spindle poison (vincristine). Even though exogenous overexpression of wild-type p53 has been associated with a chemosensitive phenotype in several model systems we demonstrated no such relationship in these studies. In addition, expression levels of Bcl-2, Bcl-xL, Bcl-xS, Bak, or Mcl-1 did not correlate with sensitivity or resistance to the four drugs. However, there was a statistically significant correlation between endogenous levels of Bax protein and sensitivity to both doxorubicin and actinomycin D. We conclude that even though many proteins such as p53 and Bcl-2 have been shown to influence drug response when exogenously overexpressed in model systems, in unmodified cell lines endogenous protein levels may not generate the same results. We have demonstrated that endogenous Bax expression was the only protein found to be associated with chemosensitivity across the three different tumor histiotypes and propose that analysis of Bax may be a more useful prognostic indicator for tumor response to therapy than either p53 or Bcl-2.