Dosimetric Comparison of Craniospinal Irradiation Using Different Tomotherapy Techniques.

The objective of this study is to compare the new and conventional tomotherapy treatment techniques and to evaluate dosimetric differences between them. A dosimetric analysis was performed by comparing planning target volume (PTV) median dose, 95% of PTV dose coverage, Paddick conformity index (CI), homogeneity index (HI), whole-body integral dose, and OAR median doses. The beam on time (BOT) and the effect of different jaw sizes and pitch values was studied. The study results indicated that the PTV dose coverage for all the techniques was comparable. Treatment plans using dynamic jaw reduced OAR doses to structures located at the treatment field edge compared to fixed jaw plans. The HT-3DCRT plans resulted in higher OAR doses to kidney, liver, and lung compared to the other techniques, and TD-IMRT provided the best dose sparing to liver compared to other techniques. Whole-body integral dose differences were found to be insignificant among the techniques. BOT was found to be higher for fixed jaw treatment plan compared to dynamic jaw plan and comparable between all treatment techniques with 5-cm dynamic jaw. In studying effect of jaw size, better OAR sparing and HI were found for 2.5-cm jaw but at the expense of doubling of BOT as compared to 5-cm jaw. There was no significant improvement found in OAR sparing when the pitch value was increased. Increasing the pitch from 0.2 to 0.43, the CI was improved, HI improved only for 5-cm jaw size, and BOT decreased to approximately half of its original time.

Clinical feasibility of TBI with helical tomotherapy.

Our purpose was to present the clinical feasibility of TBI with helical tomotherapy (HT) in four patients with AML. Treatment planning, delivery, dose verification and summation, toxicity and patient outcomes for each patient are presented. TBI prescription was set in such a manner that 80% of the clinical target volume received 12 Gy in six fractions, at two fractions per day. Dose reconstruction was carried out by recontouring the regions of interest in the daily pretreatment megavoltage computed tomography of each individual fraction and calculating its corresponding dose. A deformable registration model was used for dose summation of all individual fractions. Differences between planned and delivered doses were calculated. Average planned and delivered doses to the regions of interest differed by up to 2.7%. TBI toxicity was limited to radiotherapy oncology group grade 1 dermatitis in all patients and grade 1 headache in one patient. Two patients are alive with no evidence of disease and no GVHD. Two patients died of GVHD, but there was no evidence of disease at the time of death. We conclude that HT simplifies the process of TBI. Dose verification is possible with HT showing small differences between plan and delivered doses.

Replicating adenoviral vector-mediated transfer of a heat-inducible double suicide gene for gene therapy.

Tumor cells that express a fusion gene of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (TK) sequences activate and are subsequently killed by the nontoxic prodrugs 5-fluorocytosine and ganciclovir. We have previously developed a recombinant adenovirus containing the CD-TK fusion gene controlled by the human inducible heat shock protein 70 promoter so that heat at 41 degrees C for 1 hour induces therapeutic gene expression. This adenovirus effectively transduces heat-inducible expression of the CD-TK gene into human prostate carcinoma cells. However, because a limited number of cells in a tumor can actually be infected, we created a replicating adenoviral vector to increase CD-TK gene expression. This vector is a replication-competent, E1B-attenuated adenoviral vector containing the hsp70 promoter-driven CD-TK gene (Ad.E1A(+)HS-CDTK). When human prostate adenocarcinoma DU-145 cells (mutant p53) were infected with the virus at a multiplicity of infection (MOI) of 1 or 10, the viral replication was detected within 2 days at both MOIs. Similar results were observed in human colorectal carcinoma CX-1 cells. When DU-145 cells were infected with the virus at an MOI of 10, incubated for 24 hours, heated at 41 degrees C for 4 hours, and then harvested 20 hours later, Western blot analysis demonstrated that this virus successfully produced viral E1A proteins and heat shock stimulated the CD-TK gene expression by 12.3-fold. In addition, Ad.E1A(+)HS-CDTK effectively suppressed cell proliferation by viral cytopathic effect). Unlike with a replication-incompetent virus (Ad.HS-CDTK), the cytopathic effect of the virus and cytotoxicity in the presence of the prodrugs were still observed even at low MOI (MOI=1.0).

Heat-activated transgene expression from adenovirus vectors infected into human prostate cancer cells.

Replication-deficient adenovirus expression vectors were used to introduce a recombinant DNA construct containing enhanced green fluorescent protein (EGFP) under control of a truncated, human heat shock promoter into human prostate cancer cells growing either exponentially or in plateau phase. This was done to measure controlled, heat shock-induced EGFP expression under conditions relevant to treating human cancers with heat-activated gene therapy. Both the temporal duration and magnitude of EGFP expression increased proportionately with stronger heat shocks (time at temperature) up to maximum values that were induced by 4 h at 41.0 degrees C or 2 h at 42.0 degrees C. Longer heat shocks at either temperature yielded no additional EGFP expression and ultimately reduced it. Maximal EGFP expression was induced in exponential cultures by heat shocks delivered 12-24 h after virus infection. Induction at progressively later postinfection times induced increasingly lower, peak EGFP expression. Maximal EGFP expression could not be induced until 48 h after infection of plateau phase cultures but could still be induced 180 h after virus infection. However, peak EGFP levels in plateau cultures were approximately 25-50% of those observed in identically induced exponential cultures. Ostensibly, the differences in expression from the heat shock promoter observed in exponential and plateau cultures were attributable to cell division diluting the vector within exponential cultures and the lower metabolic activity in serum-starved plateau cultures. For all experimental conditions, EGFP expression induced from the heat shock promoter was comparable with or higher than that from the constitutively active cytomegalovirus promoter over any 24-h period. The experimental results demonstrated that EGFP expression from the heat shock promoter was controllable in both exponential and plateau phase cultures and support the plausibility of using controlled heat shock activation of this promoter as a means of regulating both the spatial and temporal expression of therapeutic DNA constructs within human tissues. The ability to localize and regulate expression from the heat shock promoter may prove particularly advantageous for many cancer applications, especially if the therapeutic products are highly toxic, e.g., proteotoxins or cytokines. However, the results of this study suggest that differential growth conditions within tumors could markedly affect the expression of recombinant DNA under control of both inducible and constitutive promoters. Consequently, inducing schemes may need to be spatially adjusted to obtain the desired therapeutic results in all tumor domains using heat-activated gene therapy.

Dominant-negative Jun N-terminal protein kinase (JNK-1) inhibits metabolic oxidative stress during glucose deprivation in a human breast carcinoma cell line.

Signal transduction pathway involved in glucose deprivation-induced oxidative stress were investigated in human breast carcinoma cells (MCF-7/ADR). In MCF-7/ADR, glucose deprivation-induced prolonged activation of c-Jun N-terminal kinase (JNK1) as well as cytoxicity and the accumulation of oxidized glutathione. Glucose deprivation also caused significant increases in total glutathione, cysteine, gamma-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of gamma-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of gamma-glutamylcysteine synthetase, suggesting that the synthesis of glutathione increased as an adaptive response. Expression of a catalytically inactive dominant negative JNK1 in MCF-7/ADR inhibited glucose deprivation- induced cell death and the accumulation of oxidized glutathione as well as altered the duration of JNK activation from persistent (> 2 h) to transient (30 min). In addition, stimulation of glutathione synthesis during glucose deprivation was not observed in cells expressing the highest levels of dominant negative protein. Finally, a linear dose response suppression of oxidized glutathione accumulation was noted for clones expressing increasing levels of dominant negative JNK1 during glucose deprivation. These results show that expression of a dominant negative JNK1 protein was capable of suppressing persistent JNK activation as well as oxidative stress and cytotoxicity caused by glucose deprivation in MCF-7/ADR. These findings support the hypothesis that JNK signaling pathways may control the expression of proteins contributing to cell death mediated by metabolic oxidative stress during glucose deprivation. Finally, these results support the concept that JNK signaling-induced shifts in oxidative metabolism may provide a general mechanism for understanding the diverse biological effects seen during the activation of JNK signaling cascades.

Adenoviral transduction of a cytosine deaminase/thymidine kinase fusion gene into prostate carcinoma cells enhances prodrug and radiation sensitivity.

Prostate tumor cells (PC-3) were transduced with defective, recombinant adenovirus containing a fusion gene encoding the Escherichia coli cytosine deaminase and herpes simplex virus type-1 thymidine kinase under the control of a cytomegalovirus promoter. Expression levels of the fusion protein were dependent on the multiplicity of infection used and incubation time following infection. PC-3 cells expressing this protein were sensitized to killing by the normally innocuous prodrugs 5-fluorocytosine and ganciclovir. In addition, radiation-induced killing was enhanced in virally infected cells in the presence of the prodrugs.

Metabolic oxidative stress activates signal transduction and gene expression during glucose deprivation in human tumor cells.

The mechanism of glucose deprivation-induced activation of Lyn kinase (Lyn), c-Jun N-terminal kinase 1 (JNK1) and increased expression of basic fibroblast growth factor (bFGF) and c-Myc was investigated in MCF-7/ADR adriamycin-resistant human breast carcinoma cells. Glucose deprivation significantly increased steady state levels of oxidized glutathione content (GSSG) and intracellular prooxidants (presumably hydroperoxides) as well as caused the activation of Lyn, JNK1, and the accumulation of bFGF and c-Myc mRNA. The suppression of GSSG accumulation and prooxidant production by treatment with the thiol antioxidant, N-acetylcysteine, also suppressed all the increases in kinase activation and gene expression observed during glucose deprivation. In addition, glucose deprivation was shown to induce oxidative stress in IMR90 SV40 transformed human fibroblasts, indicating that this phenomena is not limited to the MCF-7/ADR cell line. These and previous observations from our laboratory show that glucose deprivation-induced oxidative stress in MCF-7/ADR cells activates signal transduction involving Lyn, JNK1, and mitogen activated protein kinases (ERK1/ERK2) which results in increased bFGF and c-Myc mRNA accumulation. These results provide support for the hypothesis that alterations in intracellular oxidation/reduction reactions link changes in glycolytic metabolism to signal transduction and gene expression in these human tumor cells.

Hypoxia-induced bFGF gene expression is mediated through the JNK signal transduction pathway.

Although the synthesis of angiogenic factors in hypoxic regions of solid tumors is recognized as one of the critical steps in tumor growth and metastasis, the signal transduction pathway involved in hypoxic induction of basic fibroblast growth factor (bFGF) gene expression is still obscure. In the study described here, we investigated the intracellular responses to hypoxia and the mechanisms triggering the initiation of angiogenic activity in drug-resistant human breast carcinoma MCF-7/ADR cells. Northern blots showed an increase in the level of c-jun, c-fos, and bFGF mRNA during hypoxia. Gel mobility-shift analysis of nuclear extracts from hypoxia-exposed cells showed an increase in AP-1 binding activity. In addition, hypoxic treatment strongly activated c-Jun N-terminal kinase 1 (JNK1), leading to phosphorylation and activation of c-Jun. Expression of a dominant negative mutant of JNK1 suppressed hypoxia-induced JNK1 activation as well as bFGF gene expression. Taken together, hypoxia-induced bFGF gene expression is mediated through the stress-activated protein kinase (SAPK) signal transduction pathway.

Diamide-induced cytotoxicity and thermotolerance in CHO cells.

Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide-denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0 degrees C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees C [Freeman et al., J. Cell. Physiol., 164:356-366 (1995); Senisterra et al., Biochemistry 36: 11002-11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.

Metabolic oxidative stress-induced HSP70 gene expression is mediated through SAPK pathway. Role of Bcl-2 and c-Jun NH2-terminal kinase.

In previous reports we demonstrated that glucose deprivation induces metabolic oxidative stress in drug-resistant human breast carcinoma MCF-7/ADR cells (Lee, Y. J., Galoforo, S. S., Berns, c. M., Chen, J. C., Davis, B. H., Swim, J. E., Corry, P. M., and Spitz, D. R. (1998) J. Biol. Chem. 273, 5294-5299). In the study described here, we investigated intracellular responses to metabolic oxidative stress. Northern blots show an increase in the level of HSP70 and HSP28 mRNA in cells exposed to glucose-free medium for 1 h. One- and two-dimensional polyacrylamide gel analyses confirmed that glucose deprivation induced a family of HSPs, particularly an inducible HSP70. Overexpression of bcl-2 suppressed glucose deprivation-induced HSP70 gene expression, heat shock transcription factor-heat shock element binding activity, as well as c-Jun NH2-terminal kinase (JNK1) activation. Expression of a dominant-negative mutant of JNK1 also suppressed glucose deprivation-induced JNK1 activation as well as HSP70 gene expression. Taken together, the stress-activated protein kinase signal transduction pathway is involved in glucose deprivation-induced heat shock gene expression.

Adenoviral-mediated transfer of a heat-inducible double suicide gene into prostate carcinoma cells.

Tumor cells that express a fusion gene comprised of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (TK) sequences exhibit activation of and subsequent killing by the normally innocuous prodrugs 5-fluorocytosine and ganciclovir (Rogulski et al., Hum. Gene Ther., 8: 73-85, 1997). To target localized expression of this therapeutic gene, we have constructed a recombinant adenovirus containing the CD-TK fusion gene under the control of a human inducible heat shock protein 70 promotional sequence. Strong expression of the fusion gene product was induced by heating at 41 degrees C for 1 h. Expression levels obtained were dependent on the multiplicity of infection used and the incubation time after heat shock. Heat-induced expression of the CD-TK protein significantly reduced the survival of PC-3 cells in the presence of both 5-fluorocytosine and ganciclovir. These studies represent a novel form of gene therapy for the transduction and regulation of a double suicide gene in tumor cells and may provide a unique application for hyperthermia in cancer therapy.

Differential induction of cell death in human glioma cell lines by sodium nitroprusside.

BACKGROUND: High grade gliomas represent very aggressive and lethal forms of human cancer, which often exhibit recurrence after surgical intervention and resistance to conventional chemotherapeutic and radiologic treatment. The clinically approved antihypertensive agent sodium nitroprusside (SNP) has been shown to induce cytotoxicity toward a number of carcinoma cell lines in vitro. METHODS: Three human glioma cell lines were examined for susceptibility to the cytotoxic effects of SNP. The role of the protein kinase C (PKC)alpha gene in mediating resistance to SNP-induced killing in U343 cells was investigated using antisense oligonucleotide inhibition. Stable transfection and overexpression of the PKCalpha gene in the SNP-susceptible cell line U251 was performed to further implicate PKCalpha as a mediating factor in SNP cytotoxicity. In addition, the presence of bcl-2 protein in these cells was examined for possible correlation(s) with resistance to SNP. RESULTS: Exposure of U251 cells and LN-Z308 cells to 0.5 mM SNP resulted in significant cytotoxicity over a 72-hour period. U343 cells were resistant to SNP killing. U343 cells were shown to exhibit higher basal levels of PKCalpha and bcl-2 than either U251 or LN-Z308 cells. bcl-2 expression and resistance to SNP toxicity both were decreased by the introduction of PKCalpha antisense oligonucleotides into U343 cells. Conversely, enhanced PKC activity in PKCalpha-transfected U251 clones was associated with increased bcl-2 expression and greater resistance to SNP-induced toxicity relative to control transfected cells. CONCLUSIONS: SNP can induce cytotoxicity in glioma cells. The susceptibility of these glioma cells to nitroprusside-induced killing appears to be correlated inversely with bcl-2 and PKC activity. bcl-2 levels in these cells can be altered through modulation of PKC signaling, specifically, by induction or inhibition of PKCalpha. These in vitro results provide an interesting basis for further study into the potential use of SNP for treatment of human gliomas in patients receiving combination therapy with conventional chemotherapeutic agents that exhibit PKC inhibitory activity.

Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells.

We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.

Comparison of tumor growth between hsp25- and hsp27-transfected murine L929 cells in nude mice.

We have developed a novel system for examining the possible contribution of small heat shock proteins (hsp) to tumor growth. L929 fibrosarcoma cells, which do not express significant levels of endogenous hsp25, were stably transfected with either murine hsp25 or human hsp27. Both transfected genes were over-expressed and the respective proteins were phosphorylated in L929 cells. L929 cells transfected with hsp25 exhibited enhanced tumor growth compared to control transfected L929 cells upon s.c. injection into nude mice. In contrast, cells transfected with hsp27 exhibited delayed tumor progression in comparison to controls. Although these 2 heat shock genes and respective proteins are structurally very similar, they apparently exhibit distinct effects on tumor growth in this system.

Pulsed low dose rate brachytherapy in a rat model: dependence of late rectal injury on radiation pulse size.

PURPOSE: Clinical protocols utilizing pulsed low dose rate brachytherapy (PDR) to replace traditional continuous low dose rate brachytherapy (CLDR) employ irradiation in individual pulses given at intervals of a few hours. A critical factor in determining whether PDR will produce equivalent or greater late-occurring normal tissue toxicity is the dose per pulse. A rat rectal model was used to determine the role of pulse size in modifying dose effectiveness in producing late-occurring toxicity. METHODS AND MATERIALS: A rat model in which the rectum is irradiated with 192Ir sources was used in conjunction with an intracavitary applicator. A section of rectum 1.3 cm in length was irradiated with either 0.75 Gy/h CLDR or one of five schemes of PDR. The schemes applied 0.375, 0.75, 1.5, 3.0, or 6.0 Gy pulses at 0.5, 1.0, 2.0, 4.0, or 8.0 h intervals, respectively. Rats were observed for up to 300 days after completion of irradiation for rectal obstruction. Rectal specimens were taken at the time of sacrifice for obstruction or at the end of follow-up and analyzed histologically for injury. RESULTS: Effectiveness of irradiation was analyzed by calculating the ED50 for incidence of obstruction and severe histological injury. The ED50 for obstruction after treatment with CLDR and pulse sizes of 0.375, 0.75, and 1.5 Gy were 70.5, 68.0, 68.6, and 68.8 Gy, respectively. These values were not significantly different. Compared to CLDR, the ED50 for obstruction after pulse sizes of 3.0 and 6.0 Gy were significantly different at 60.9 and 46.3 Gy, respectively. The relative changes in ED50 for the different radiation schemes in producing ulceration, fibrosis, and vascular sclerosis injury were similar to that observed for obstruction. The endpoints of colitis cystica profunda and atypical epithelial regeneration varied less with increasing pulse size. CONCLUSIONS: We have demonstrated that for late rat rectal injury, dose responses to PDR pulse sizes up to 1.5 Gy at 2-h intervals are not distinguishable from that seen with CLDR at a dose rate of 0.75 Gy/h.

Hypoglycemia-induced c-Jun phosphorylation is mediated by c-Jun N-terminal kinase 1 and Lyn kinase in drug-resistant human breast carcinoma MCF-7/ADR cells.

We studied the signal transduction mechanism that is involved in c-Jun phosphorylation evident after glucose deprivation in MCF-7/ADR cells. Glucose deprivation caused an immediate increase in tyrosine phosphorylation in MCF-7/ADR cells and specifically activated Lyn kinase, a src family tyrosine kinase. In addition, hypoglycemic treatment strongly activated c-Jun N-terminal kinase 1 (JNK1), leading to the phosphorylation and activation of c-Jun. Experiments with Lyn antisense oligonucleotides demonstrated that Lyn kinase activation was responsible for the activation of JNK1 but not extracellular signal-regulated kinase. We also observed glucose deprivation-induced Ras activation in MCF-7/ADR cells. These results indicate a possible Ras-dependent signaling pathway involving Lyn kinase and JNK1, which leads to the glucose deprivation-induced responses in MCF-7/ADR cells.

Differential effect of glucose deprivation on MAPK activation in drug sensitive human breast carcinoma MCF-7 and multidrug resistant MCF-7/ADR cells.

We have investigated the effect of glucose deprivation treatment on the activation of mitogen activated protein kinases (MAPKs) in the drug-sensitive human breast carcinoma cells (MCF-7) and its drug resistant variant (MCF-7/ADR) cells. Western blots and in-gel kinase assays showed that glucose free medium was a strong stimulus for the activation of MAPK in MCF-7/ADR cells. No activation was seen in MCF-7 cells. MAPK was activated within 3 min of being in glucose free medium and it remained activated for over 1 h in MCF-7/ADR cells. After being returned to complete medium, 1 h was required for the MAPK to become deactivated. To investigate whether alternative sources of ATP could inhibit glucose deprivation induced MAPK activation, we added glutamine and glutamate to glucose deprived medium. The addition of glutamine did not reverse glucose deprivation induced MAPK activation in MCF-7/ADR cells. The addition of glutamate, however, decreased the MAPK activation and the length of time of activation. We observed an increase greater than three fold in MEK, Raf, Ras, and PKC activity with glucose deprivation in MCF-7/ADR cells. This suggests that glucose deprivation-induced MAPK activation is mediated through this signal transduction pathway.

Examination of the molecular basis for the lack of alphaB-crystallin expression in L929 cells.

We have previously shown that murine L929 cells do not express the small heat shock protein alphaB-crystallin upon exposure to thermal stress (Mol Cell Biochem 155: 51-60, 1996). In these studies, we demonstrate that L929 cells also fail to express alphaB-crystallin upon exposure dexamethasone, whereas NIH 3T3 and Swiss 3T3 murine cells exhibit alphaB-crystallin expression under identical conditions. Mobility shift assays demonstrated heat-inducible binding, presumably by heat shock factor(s), to an alphaB-crystallin heat shock element (HSE) oligomeric sequence in total cellular extracts from L929 cells. Transient transfection of a plasmid containing the alphaB-crystallin promoter linked to a CAT reporter gene exhibited heat-inducible expression in L929 cells. In addition, L929 cells stably transfected with a plasmid containing the complete alphaB-crystallin gene showed expression of this gene following heat shock. The presence of the endogenous alphaB-crystallin gene was detected by Southern blot hybridization of genomic L929 DNA, and sequence analysis revealed identical nucleotide structure to published murine sequences throughout the entire promoter. Treatment of L929 cells with 5-azacytidine enabled heat-inducible expression of alphaB-crystallin from the endogenous gene, however, methylation of the putative heat shock element (HSE) and flanking promoter sequences of L929 cell genomic DNA was not detected. In vivo genomic footprinting demonstrated constitutive binding to the endogenous HSE of the alphaB-crystallin promoter in L929, L929/alphaB-crystallin transfectant cells, and Swiss 3T3 cells during unstressed and heat stressed conditions. Therefore, the genomic alphaB-crystallin HSE region in L929 cells appears to be available for binding of putative transcription factors, but methylation in other regions of the gene or genome repress the expression of alphaB-crystallin in L929 cells. In vitro culture of L929 cells appears to have rendered the alphaB-crystallin gene loci inactive through methylation, thus providing a unique system by which to study the function of transfected small heat shock proteins.

Reproducible rat model for comparing late rectal toxicity from different brachytherapy techniques.

PURPOSE: Newer brachytherapy techniques, such as pulse-simulated low dose rate (PDR) and high dose rate (HDR) offer clinical and technical advantages over the conventional continuous low dose rate (CLDR) irradiation. The impact of these techniques on late normal tissue toxicity has remained largely undefined, with mathematical modeling and limited clinical experience providing some guidelines. We sought to develop a reproducible rat model for quantifying and comparing late rectal toxicity from different brachytherapy techniques. METHODS AND MATERIALS: An intrarectal applicator has been designed and techniques have been developed to deliver clinically relevant doses of CLDR to allow comparison to PDR and HDR. Female Wistar rats are utilized. The endpoints assessed are rectal obstruction and a histological grading of late rectal injury. RESULTS: Analysis of 65 rats given either sham or single acute pulse irradiation has demonstrated the model to be reproducible and reliable in evaluating late rectal toxicity. CONCLUSIONS: The use of this rat model to compare late rectal injury from various doses, dose rates, and fractionation parameters utilized by CLDR, PDR, and HDR will provide another tool for safely implementing these techniques into the clinical setting.