The current study was undertaken to investigate the influence of wild-type or mutant p53 status on the radiosensitizing effect of paclitaxel in colorectal tumor cell lines.
HCT-116 (contains ...wild-type p53) and HT-29 (contains mutant p53) established from moderately differentiated colorectal carcinomas were used in this study. Colony-forming assay was performed after exposure to either different radiation doses (0.5-6 gray Gy) or paclitaxel (1-10 nM) or in combination. Induction of p53 and p21(waf1/cip1) by these treatments were determined by immunocytochemistry and Western blot analysis.
Radiation caused an increase in nuclear p53 and p21(waf1/cip1) proteins in HCT-116 cells, indicating that p53 functionally induced p21(waf1/cip1). However, induction of nuclear p53 and p21(waf1/cip1) protein was not evident in HT-29 cells, suggesting that p53 was not functional in these cells. Survival data showed that the HCT-116 cells (survival fraction of exponentially growing cells that were irradiated at the clinically relevant dose of 2 Gy SF(2) = 0.383; dose required to reduce the fraction of cells to 37% D(0) = 223 centigray cGy) were significantly sensitive to ionizing radiation (P < 0.008) when compared with the HT-29 cells (SF(2) = 0.614; D(0) = 351 cGy). Paclitaxel caused a higher degree of clonogenic inhibition in HCT-116 (D(0) = 0.7 nM) than HT-29 (D(0) = 1.11 nM) cells (P < 0.06). When paclitaxel and radiation were combined, an enhanced radiosensitizing effect (P < 0.05) was observed in HCT-116 cells (SF(2) = 0.138; D(0) = 103 cGy), whereas in HT-29 cells no significant radiosensitization of paclitaxel was observed (SF(2) = 0.608; D(0) = 306 cGy). However, pretreatment with paclitaxel followed by multifractionated low dose radiation (0.5- or 1-Gy fractions for a total dose of 2 Gy) significantly enhanced the radiosensitizing effect in both HCT-116 and HT-29 cells.
The results of the current study suggested that multifractionated radiation given at very low doses after exposure of cells to paclitaxel conferred a potent radiation sensitizing effect irrespective of p53 status.
While p53 protein plays an important role in the regulation of radiosensitivity and chemosensitivity in many tumors, the role of p53 in the combined management of tumors that harbor mutations in the ...p53 gene have not been fully defined. This study was undertaken to evaluate the impact of wild-type or mutant p53 status on the synergistic effects of 5-Fluorouracil (5-FU) and radiation (XRT) in pancreatic tumors.
Three pancreatic tumor cell lines, one containing wild-type functional p53 (Capan-2) and two containing mutant p53 (Panc-1 and MIA PaCa-2), were used in this study. Radiation-induced p53 and p21(waf1/cip1) protein expression was determined by Western blot analysis. Radiation induced Thymidylate Synthase (TS) mRNA expression was determined by 32P-RT-PCR. The effect of 5-FU, radiation, and radiation +5-FU on the growth and colony-forming ability of Capan-2, Panc-1 and MIA PaCa-2 was determined by clonogenic assays respectively.
Radiation elevated p53 and p21(waf1/cip1) levels in Capan-2 cells. No elevation of p53 and p21(waf1/cip1) was evident in Panc-1. MIA PaCa-2 cells showed down-regulation of p21(waf1/cip1) with no elevation of p53 protein. Clonogenic assays showed enhanced radiosensitizing effect when 5-Fluorouracil was added to cell lines lacking functional p53. In wild-type p53 Capan-2 cells, radiation up-regulated TS mRNA levels. High basal levels of TS mRNA were detected in p53 mutant cell lines with no evident induction by radiation.
Our results confirm that p53 status has a significant impact on radiation sensitivity with wild-type p53 cells being significantly more radiosensitive than mutant cell lines. When XRT and 5-FU were combined, this led only to an additive effect in wild-type cell lines and a synergistic effect in mutant cell lines.
Objectives. To determine the extent of iatrogenic tumor cell dissemination during brachytherapy by assessing prostate-specific antigen (PSA) mRNA expression in circulating prostate tumor cells using ...reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The instrumentation used in the radioisotope seed placement of the prostate causes trauma to blood vessels and provides a vascular access for tumor cells that can lead to potential iatrogenic dissemination and systemic failure.
Methods. Twenty-five patients treated for brachytherapy were recruited in the study. Controls included 4 normal men and 1 woman; case controls included 4 patients who underwent prostate biopsy for prostate cancer diagnosis. Peripheral blood (10 mL) was collected before, during, and after the brachytherapy procedure. Total RNA was isolated from mononuclear cells and phosphorus-32 RT-PCR was performed to analyze the mRNA expression of PSA and G6PDH genes.
Results. Of 25 patients, 23 were negative for PSA mRNA expression and 2 were positive for PSA mRNA expression before brachytherapy. Of the 23 patients who were negative for PSA mRNA expression before treatment, 15 patients (65%) turned positive during or after brachytherapy and the remaining 8 patients remained negative throughout the treatment. Eight of the 25 patients developed rising serum PSA levels. Of these 8 patients, 1 (12.5%) did not have PSA mRNA expression in the peripheral blood before, during, or after brachytherapy; the remaining 7 patients who developed rising serum PSA levels had PSA mRNA expression after brachytherapy (
P = 0.03).
Conclusions. These findings strongly suggest that iatrogenic shedding of prostate cells occurs as a result of brachytherapy and raises the concern that these cells liberated at the time of brachytherapy increase the risk of metastatic deposits and results in systemic failure, as measured by serum PSA levels.
Activated ras is known to dysregulate TGF-beta signaling by altering the expression of TGF-beta type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant ...to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-beta signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 microM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 microM concentrations (P>0.004). The TGF-beta effector gene p21(waf1/cip1) was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-beta responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-beta signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT-PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-beta signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.
In this study, we sought to investigate the mechanism of the proapoptotic function of Egr -1 in relation to p53 status in normal isogenic cell backgrounds by using primary MEF cells established from ...homozygous ( Egr -1 â
/â ) and heterozygous ( Egr -1 +/â ) Egr -1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis in Egr -1 +/â cells (22.8%; p < 0.0001) when compared with Egr -1 â
/â cells (3.5%). Radiation elevated p53 protein in Egr -1 +/â cells in 3â6 h. However, in Egr- 1 â
/â cells, the p53 protein was down-regulated 1 h after radiation and was completely degraded at the later time points. Radiation
elevated the p53-CAT activity in Egr -1 +/â cells but not in Egr -1 â
/â cells. Interestingly, transient overexpression of EGR-1 in p53 â
/â MEF cells caused marginal induction of radiation-induced apoptosis when compared with p53 +/+ MEF cells. Together, these results indicate that Egr -1 may transregulate p53, and both EGR-1 and p53 functions are essential to mediate radiation-induced apoptosis. Rb, an Egr -1 target gene, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including
hypophosphorylated forms were observed in Egr-1
â
/â MEF cells before and after radiation when compared with the levels observed in Egr-1
+/â cells. Elevated amounts of the p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed in Egr-1
â
/â cells after radiation. Because of a reduction in Rb binding to MDM2 and an increase in MDM2 binding with p53, p53 is directly
degraded by MDM2, and this leads to inactivation of the p53-mediated apoptotic pathway in Egr-1
â
/â MEF cells. Thus, the proapoptotic function of Egr -1 may involve the mediation of Rb protein that is essential to overcome the antiapoptotic function of MDM2 on p53.
In this study, we investigated whether lack of transforming growth factor β (TGF-β) type II receptor (RII) expression and
loss of TGF-β signaling played a role in radiation resistance of ...pancreatic cancer cells MIA PaCa-2 that possess a mutated p53 gene. Transfection of this cell line with a RII cDNA led to a stimulation of the transcriptional activity of p3TP-Lux, a
TGF-βâresponsive reporter construct. The RII transfectants (MIA PaCa-2/RII) showed a significant increase in sensitivity to
radiation when compared with MIA PaCa-2/vector cells. The increase in sensitivity to radiation was reversed by neutralizing
antibodies to TGF-β, indicating that these changes were dependent on TGF-β signaling. Compared with MIA PaCa-2/vector cells,
MIA PaCa-2/RII cells showed a greater than 3-fold increase in apoptosis after radiation. Enhanced radiation sensitivity of
MIA PaCa-2/RII cells was associated with an induction of Bax mRNA and protein that was followed by a release of cytochrome
c and activation of caspase-3 and poly(ADP-ribose) polymerase cleavage after radiation exposure. Overexpression of Bcl-x L or treatment with antisense oligodeoxynucleotides targeted against Bax significantly inhibited radiation-induced apoptosis
in MIA PaCa-2/RII but not in MIA PaCa-2/Vector cells, suggesting that Bax induction is necessary for radiation-induced TGF-β
signaling-mediated apoptosis. Thus, restoration of TGF-β signaling sensitized these cells to ionizing radiation, although
these cells possess a mutated p53 gene. In addition, disruption of RII function by dominant negative mutant of RII inhibited the radiation-induced TGF-β signaling
and apoptosis in primary cultures of mouse embryonic fibroblasts. Together, these observations imply that RII is an important
component of radiation-induced TGF-β signaling, and loss of function of RII may enhance resistance to radiation-induced apoptosis.
This study was undertaken to determine whether the transcription factor EGR-1 expression: (1) in the primary tumor, correlates with radiation response in terms of complete local tumor control with no ...evidence of disease or recurrence and no evidence of metastasis; (2) in the postirradiated biopsies correlates with residual tumor; and (3) correlates with the expression of Egr-1 target genes such as TP53, pRB, and Bax. The authors analyzed: (1) 25 pretreated surgically resected paraffin-embedded primary adenocarcinomas of the prostate for the presence of EGR-1 expression and mutation, and correlated this with clinical endpoints such as serum prostate-specific antigen levels and current clinical status; (2) 27 postirradiated biopsies of prostate for the presence of EGR-1 expression, and correlated these findings to the residual tumor status; and (3) 12 prospective prostate tumor specimens for EGR-1 expression and its target genes. EGR-1 expression was determined by immunohistochemistry and mutations were screened in two regions of the Egr-1 gene (trinucleotide AGC repeats in transactivation domain TD and poly A tract in 3'UTR) by polymerase chain reaction-single strand conformational polymorphism analysis. Of 25 patients, 18 patients showed expression of EGR-1. EGR-1 overexpression correlated with treatment failure. No correlation with EGR-1 overexpression and its target genes was found, which may indirectly suggest that overexpressed EGR-1 may lack transactivation function. In summary, EGR-1 overexpression in the mutant form may provide an indication of clinical failure (local recurrence or metastasis).
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