.JPG?ad=18503&adview=true)
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Biology and Genetics |
BioServe Biotechnologies [T. A. L., L. R. B., R. M.], Laurel, Maryland 20707; Departments of Therapeutic Radiology [B. G. H.] and Surgery [A. A. G.], Yale University School of Medicine, New Haven, Connecticut 06520; Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892 [S. K., P. G. S.]; and Department of Radiation Oncology and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5097 [C. J. C., B. C. T.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
p53 intronic base substitutions have been noted in patients including a p53 G to C transversion at base 13964 (p53 13964GC) in women diagnosed with both breast and ovarian cancer, but this base change has previously been thought to represent a polymorphism of unclear significance (12) . This is an important because these base changes may results in functional mutations distinction and women with LFS have an 80% lifetime risk of developing breast cancer (10) . The clinical significance of germ-line p53 gene mutations in familial breast cancer has not been considered important because less than 1% of all breast cancer patients have germ-line p53 mutations; this percentage is small compared to those with BRCA1 and BRCA2 mutations (13, 14, 15, 16, 17) .
There is accumulating evidence that novel mechanisms of gene regulation, including mutations in splice donor and acceptor sites, enhancer, intron, and promoter elements, may be important in regulating gene expression, including p53 (18 , 19) . The identification of these unique mutations that result in aberrant gene expression may provide for the detection of patients at risk for the development of cancer. The identification of these mutations may someday allow physicians to optimize treatment because these genetic changes may alter specific biochemical pathways. We now provide evidence that the p53 13964 base is mutated in patients who have familial breast carcinoma but not sporadic breast cancer. Furthermore, this mutation inhibits apoptosis and prolongs cell survival following DNA damage and thus may indeed affect breast cancer risk. The identification of women at risk for the development of breast cancer will have important implications for the prevention of cancers, treatment strategies, and improved cure rates of these patients.
|
PATIENTS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. These patients were all treated with radiation therapy and closely
followed in the clinic at Yale University School of Medicine. A
protocol for the study was approved by the Human Investigations
Committee at the Yale University School of Medicine, and all patients
signed a written informed consent form. Samples were coded and
entered into a double-blinded database. The enrolled familial breast
cancer patients underwent an extensive interview for complete family
history with a three-generation pedigree profile. After the interview,
all enrolled patients underwent standard sterile phlebotomy, from which
10–15 ml of blood was collected for genomic DNA isolation from
lymphocytes.
|
p53 and BRCA1/BRCA2 Mutations.
PCR direct sequencing was performed on all samples with mobility shifts
as described previously (21)
. Also, each putative mutant
sample was amplified and cloned using the TA cloning system. DNA from
individual colonies was isolated, and automated sequencing was
performed on a ABI377 sequencer using the M13 primer. Comprehensive
scanning of exons 2–11 of the p53 gene also was performed
with a p53 gene chip using gene array analysis (Affymetrix,
Santa Clara, CA) as described previously (22)
.
BRCA1 and BRCA2 comprehensive automated
sequencing was performed as described previously (23)
.
Genotype Detection.
As a rapid screening method for the p53 intron 6
13964GC mutation, we developed a PCR-RFLP assay.
The intron mutation site of interest was amplified using 20 pmol each
of the exon 7 PCR-SSCP primers (E7AF, 5'-CTT GCC ACA GGT CTC CCC AA-3';
E7AR, 5'-TGT GCA GGG TGG CAA GTG GC-3'). PCR was performed with 100 ng
of genomic DNA containing 20 mM Tris-HCl (pH
8.4), 50 mM KCl, 2.0 mM
MgCl2, 37.5 µM each
nucleotide, and 1.25 units of Taq polymerase (Life Technologies, Inc.).
The cycling conditions were 94°C for 5 min, followed by 35 cycles of
94°C for 1 min, 60°C for 1 min, and 78°C for 1 min, with a final
cycle of 78°C for 7 min. A 10-µl aliquot of each successful
reaction was digested with 10 U of HhaI restriction enzyme
(New England Biolabs) in 2.5 µl of 10x NEB4 with 12.5 µl of water
at 37°C for 2 h. The HhaI-digested PCR fragments were
separated on a 4% gel (3% NuSieve agarose/1% agarose). The G to C
mutation at position 13964 destroys a HhaI restriction site
within the 196-bp amplification fragment. The three possible genotypes
are defined by three distinct banding patterns: G/G (wild-type),
172 + 24 bp (complete digestion); G/C (heterozygous),
196 + 172 + 24 bp; C/C (homozygous mutant), 196 bp.
Apoptosis and Cell Survival.
Lymphoblastoid cells were obtained from patients, immortalized using
EBV and maintained in RPMI 1640 with 10% FCS and antibiotics. Survival
of cells following varying doses of cisplatinum (0, 5, 10, or 20
µg/ml) was determined by the exclusion of trypan blue dye. The human
immortalized lymphoblastoid cell lines VDS and 012 were found to
contain wild-type p53 sequences and used as controls
(24)
. Immortalized lymphoblastoid cell lines were treated
with 10 µg/ml cisplatinum for 48–72 h for maximal induction of
apoptosis. Cells were fixed in a 50:50 mix of ice-cold methanol/acetone
and stained with bis-benzimide Hoechst stain No. 33258 (Sigma Chemical
Co., St. Louis, MO) at a concentration of 16 µg/ml in PBS. Apoptotic
cells were counted by two independent observers and confirmed by
sub-G0 peak and end-labeling TUNEL
staining. Transfections of VDS, BT-16, and BT-102 cells with the
pC53-SN3 plasmid containing wild-type p53 (B. Vogelstein,
Johns Hopkins University) using LipofectAMINE (Life Technologies, Inc.)
was performed (25)
. Apoptosis in response to 10 µg/ml
cisplatinum was determined for all immortalized cell lines and stably
transfected cell lines.
Immunohistochemistry for Somatic p53 Mutations.
Following identification of the patients with the p53 intron
6 13964GC base change, the individual 10%
formalin-fixed and paraffin-embedded blocks were processed for
immunohistochemical staining as described previously (26)
.
After blocking 5-µm sections with goat suppressor serum, p53 protein
levels were determined by using the DO1 monoclonal antibody (Oncogene
Science, Uniondale, NY) to mutant p53 protein at a 1:1000 dilution.
Positive staining was defined by nuclear staining only within the
invasive breast cancer component. No antigen retrieval was used to
amplify the immunohistochemical signal. All samples were stained with
positive and negative controls that included staining of tumors with
known p53 mutations and absence of primary p53
antibody.
Statistical Analysis.
The frequency of base changes in the p53 alleles between the
patients with familial and sporadic breast cancer were compared using
the Fischer’s exact test. Differences in categorical variables between
the patient groups has been compared using the Spearman
2 test. A P of less than or equal
to 0.05 (two-tailed) was considered to indicate statistical
significance.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Germ-line p53 Mutation Analysis.
Previous studies have determined that the frequency of germ-line
p53 mutations in familial breast cancer patients is 1% or
less, but many of these reports have not investigated the importance of
intron base changes in the p53 gene (13, 14, 15, 16, 17)
.
Therefore, we investigated the frequency of both exon and intron
germ-line p53 base changes in 42 breast cancer patients with
a strong family history of breast cancer. Purified DNA obtained from
the 42 index cases was screened for germ-line p53 mutations
in exons 2–11 and surrounding introns using a combination of intron
based primers for PCR-SSCP analysis, direct sequencing, and microarray
sequencing using the Affymetrix p53 gene chip methodology.
We identified two samples (BT-16 and BT-31) that had intron 6/exon
7/intron 7 gel shift alterations (Fig. 1A
,lanes 2 and 10) and a single sample (BT-102)
that had a homozygous gel shift alteration by SSCP (Fig. 1A
, lane
13). Because examination of exon 7 and the adjoining splice sites
revealed no mutation by direct sequencing, we concentrated on
sequencing all of the intron 6 and intron 7 regions that were present
in the PCR-SSCP fragment. We identified a p53
13964GC intron 6 mutation by direct sequencing
which was verified by cloning the PCR products (TA cloning system) and
sequencing. We determined that BT-16 and BT-31 were heterozygous (3 of
6 and 4 of 10 clones were mutants), and BT-102 was homozygous
for the p53 13964GC base change by
automated sequencing (9 of 9 clones were mutants; Fig. 1B
).
Comprehensive scanning of p53 exons 2–11 was performed by
gene array analysis (Affymetrix) and revealed no missense mutations in
exons 2–11 of the p53 gene in any of the 42 familial breast
cancer samples.
|
. Using the genotyping assay, we screened the 42 familial breast cancer
patients and identified only those patients previously found on SSCP
and sequencing to have the heterozygous (BT-16 and BT-31) and
homozygous (BT-102) mutations at base 13964 of the p53 gene
(Fig. 2)
. Two samples (BT-36 and BT-52) that contained
p53 wild-type sequences were used as controls and produced
the expected 172- and 24-bp fragments on HhaI digestion
(Fig. 2)
.
|
. The sporadic and familial breast cancer cohorts were found
to be matched for histopathology (adenocarcinoma), stage (I/II), and
Ashkenazi ancestry as determined by 2 analysis
(Table 1)
. We found the age of presentation of familial breast cancer
patients to be 44 years compared to 55 years for sporadic breast cancer
patients (P < 0.001; Table 1
). We identified
0 of 171 sporadic breast cancer patients to have the p53
13964GC base change compared to 3 of 42 (7%)
index cases (Fisher’s exact test, P = 0.0003). Thus, there was a highly statistically significant association
between the intron 6 p53 13964GC
mutation and familial breast cancer.
Clinical and Molecular Characteristics of Patients with the
p53 13964GC Base Change.
The ages of presentation of the three patients were 30, 42, and 59
years; all three patients presented with stage I or II invasive ductal
carcinoma, and none developed metastatic disease (Table 2)
. We found that both patients with the heterozygous p53
13964GC base change had a family history of pre-
and post-menopausal breast cancers and that both had bilateral
breast cancer consistent with the inheritance of a dominant gene
(30)
. Interestingly, both of these patients were of
Ashkenazi ancestry, although no previously defined relationship between
p53 gene mutations and Ashkenazi ancestry has been
described. We were able to obtain the paraffin-embedded breast tumor
blocks from a proband’s (BT-16) mother and maternal aunt, who had a
history of breast cancer diagnosed at ages 42 and 55, respectively.
Genotyping revealed the p53 13964GC
base change in both affected relatives, demonstrating that the genetic
change appears to track with the cancer-prone family members. Tissue
from other family members to evaluate for the p53
13964GC mutation was not available. The single
patient with the homozygous p53
13964GC mutation has a strong family cancer
history, including postmenopausal breast cancers, early and late onset
leukemias, brain tumors, and lung cancers from both maternal and
paternal lineages with no evidence of consanguinity within this family
(Fig. 3)
. We performed complete sequencing of all coding exons and intron/exon
junctions of BRCA1/BRCA2 genes in these patients and found
all patients with the p53 13964GC
mutation to have wild-type BRCA1/BRCA2 genes.
|
|
. The survival of BT-16 and BT-102 cells at 48 h
following treatment with 5, 10, and 20 µg/ml cisplatinum was
79 ± 2, 75 ± 2, and 62 ± 1% for BT-16 and 89 ± 2, 82 ± 2, and 76 ± 2% for BT-102, respectively, compared to
42 ± 3, 31 ± 3, and 18 ± 2% for the p53 wild-type lymphoblastoid cell line
VDS (Fig. 4A)
. The survival of p53 wild-type 012
cells demonstrated similar survival characteristics to VDS cells and
generally had poor survival following chemotherapy treatment compared
to BT-16 and BT-102 cells (Fig. 4A)
. Chemotherapy-induced
apoptosis was determined in two p53 wild-type lymphoblastoid
cell lines and was found to be maximal at 48 h following drug
treatment (data not shown). Treatment of BT-16 and BT-102
lymphoblastoid cells with 10 µg/ml cisplatinum for 48 h resulted
in apoptosis in 15 ± 3 and 10 ± 3%,
respectively. In comparison, p53 wild-type cells VDS and 012
(data not shown) demonstrated 47 ± 5 and 38 ± 5%, respectively (Fig. 4B)
. Transfection assays
with the pC53-SN3 plasmid containing wild-type p53 into VDS,
BT-16, and BT-102 cells demonstrated partial restoration of normal
chemotherapy-induced apoptosis in the BT-102 cells containing the
homozygous p53 13964GC base change but
not in BT-16 cells containing the heterozygous base alteration.
Following treatment with cisplatinum, apoptosis was found in
30 ± 4 of BT-102/p53T cells compared with only
10 ± 3% of untransfected BT-102 cells (Fig. 4B)
. The morphological changes of nuclear blebbing and
membrane fragmentation characteristic of apo-ptosis are shown by
staining of cells with bis-benzimide Hoechst stain are prominent in
p53 wild-type cell lines VDS and 012 (Fig. 5, C and D)
but poorly demonstrated in the BT-16
and BT-102 lymphoblastoid cell lines (Fig. 5, A and B)
.
|
|
We obtained the paraffin-embedded breast tumor blocks from patients
BT-16, BT-31, and BT-102, which were found to have the germ-line
p53 13964GC mutation. Immunostaining
of breast tumor specimens from BT-102 (Fig. 6A)
, BT-31 (Fig. 6B)
, and BT-16 (Fig. 6C)
revealed strong nuclear staining within the invasive
ductal component of all three patients. Previously, LFS patients with
p53 mutations have been shown to have high levels of mutant
p53 protein in benign tissue containing only the germ-line heterozygote
p53 mutation, but there was little benign tissue on these
sections, and expression of mutant p53 in benign tissue could not be
evaluated. Staining of sections with a secondary antibody alone
revealed no nuclear reactivity, demonstrating the lack of nonspecific
cross-reactive proteins (Fig. 6D)
.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In a cohort of breast cancer patients with a strong family history of breast cancer, 3 of 42 (7%) have the specific p53 13964GC mutation compared with 0 of 171 sporadic breast cancer patients (P = 0.003). A detailed analysis of the pedigrees of these patients reveals a pattern of late-onset cancers that appear to be inherited in an autosomal dominant pattern. Late-onset Li-Fraumeni cancers have been described previously in families with exon 4 mutations and indicate that some germ-line p53 mutations may allow for partial function of the protein, which results in the late-onset phenotype of tumors (19 , 34) . Genetic analysis of one of the proband’s mother and maternal aunt, both of whom developed breast cancer, demonstrated that they carried the p53 13964GC mutation. Examination of the pedigree from the patient with the homozygous 13964GC substitution is striking, revealing many early and late-onset LFS types of cancers including breast cancers. It is noteworthy that there are both maternal and paternal relatives with late onset breast cancers and other Li-Fraumeni-like cancers. Two of the breast cancer patients with the p53 13964GC mutation were of Ashkenazi ancestry, suggesting that this may represent a founder mutation.
It was important to establish whether this intron base change represented a functional mutation. Therefore, we established an EBV immortalized lymphoblastoid cell line from two patients with the p53 13964GC base change. The p53 protein is a transcription factor which is a downstream regulator of many signal transduction pathways, especially those that mediate an apoptotic response to chemotherapy and ionizing radiation. By using p53 knockout and transgenic mice that overexpress dominant negative p53, several independent groups have shown that p53 is essential for apoptosis following treatment with ionizing radiation, chemotherapy, or serum withdrawal (4 , 7) . A report from another group describes lymphoblastoid cell lines from LFS patients with heterozygous p53 germ-line mutations at codons 282 and 286 to be associated with resistance to radiation-induced apoptosis (35) . Our in vitro experiments demonstrating that the p53 13964GC mutation results in prolonged cell survival and inhibits chemotherapy-induced apoptosis is consistent with these findings.
The p53 13964GC base substitution was first reported by Buller et al. (12) , who found the p53 13964GC base change in 3 of 73 (4%) patients with both breast and ovarian cancer. The authors concluded that the p53 13964GC base change represents a nonfunctional polymorphism because there was no direct functional activity to implicate it in malignant transformation. Furthermore, a recent study examining children from Belarus with possible radiation-induced thyroid tumors demonstrated that 5 of 70 (7.1%) had the p53 13964GC base change, whereas 1 healthy child was also found to carry this base change (36) . The family history of these patients were not available, so no firm conclusions were made from this study with regard to associations with inherited cancers, although many childhood malignancies are often due to inherited genetic mutations. Other p53 intron 6 base substitutions have also been identified that may be associated with tumor development. A substitution at nucleotide 13961GA was identified in 6 children with the Li-Fraumeni spectrum of tumors, whereas only 1 of 184 healthy controls was found to have this base change (P = 0.036; Ref. 37 ). Tissue specimens from these patients were found to contain mutant p53 protein immunoreactivity in both tumor and normal tissue. Recently, investigators have identified another intron 6 germ-line base substitution 13492–13499TGins that was present in several members of a three generation Li-Fraumeni family characterized by breast cancers, medulloblastoma, and rhabdomyosarcoma but not identified in 85 normal control samples (38) . Both benign and malignant tissue from these family members demonstrated strong p53 protein immunoreactivity, but mRNA analysis did not demonstrate aberrant transcript production. Other intron 6 base substitutions have been reported that include changes at the regions of 13487–13494 (39 , 40) . It appears that intron 6 of the p53 gene is potentially a hot spot for mutation with novel mechanisms of gene regulation that appear to be important for tumor formation. The presence of a CpG-rich Alu repetitive element adjacent to the p53 13964 base may explain the frequency of mutation at this site (41) . It is has been reported previously that breast tumors from patients with familial breast cancer have elevated levels of mutant p53 protein without evidence of exon mutations, suggesting that mutations may occur at sites other than in exons, further supporting our findings (33) .
We found that breast cancer specimens from patients with the p53 13964GC mutation demonstrated strong nuclear p53 immunoreactivity, suggesting that the germ-line mutation causes overexpression of mutant p53 protein, similar to what occurs with missense mutations. Elevated levels of p53 protein in paraffin-embedded tumor specimens from a single Li-Fraumeni family with an intron 6 mutation was reported (38) . Identification of p53 mutations may be important for treatment-related decisions because previous studies demonstrated that breast cancer patients with p53 mutations have poor survival rates (31 , 32) . The outcome of patients with LFS including the novel p53 13964GC mutations we now describe treated with standard modern therapies, including surgery, radiation, and chemotherapy, has not yet been defined.
We now propose that the noncoding, p53 13964 base is mutated in familial breast cancer. Furthermore, intron 6 appears to be a critical location for mutation in patients with familial tumors of the Li-Fraumeni spectrum that do not have detectable germ-line p53 coding or splice site mutations. This group of Li-Fraumeni patients has late age of onset, which suggests that the function of p53 in these patients may only be partially impaired (19 , 34) . The mechanism by which the p53 13964GC mutation results in altered genetic signals remains to be determined. This site is located adjacent to an intron 6 CpG-rich Alu-repetitive element, which may explain the high rate of mutations at this specific region (41) . The detection of patients at risk for malignancies is critical in identifying candidates for screening and prevention programs and someday may allow optimal treatment. Genetic testing to identify these subgroups of patients at risk for familial breast cancer and to determine their respective outcome to modern treatments has only just begun. Our finding that BRCA1 and BRCA2 wild-type familial breast cancer patients have the germ-line p53 13964GC mutation is one step toward identifying genetic mutations that predispose women to breast cancer. Larger epidemiological studies are now warranted to further define the frequency of this mutation and perhaps detect other important intronic mutations.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was funded by grants from The Susan G.
Komen Breast Cancer Foundation (to B. C. T.) and the Radiology
Society of North America (to B. C. T.). 
2 To whom requests for reprints should be
addressed, at Bodine Center For Cancer Treatment, Department of
Radiation Oncology, Thomas Jefferson University, 111 South 11th Street,
Philadelphia, PA 19107-5097. Phone: (215) 955-6706; Fax:
(215) 955-0412; E-mail: Bruce.Turner{at}mail.tju.edu
3 The abbreviations used are: LFS, Li-Fraumeni
syndrome; SSCP, single strand conformation polymorphism.
Received 8/16/99. Accepted 12/15/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  K. Chen, Z. Hu, L.-E Wang, W. Zhang, A. K. El-Naggar, E. M. Sturgis, and Q. Wei Polymorphic TP53BP1 and TP53 Gene Interactions Associated with Risk of Squamous Cell Carcinoma of the Head and Neck Clin. Cancer Res., July 15, 2007; 13(14): 4300 - 4305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Rohan, S.-Q. Li, R. Hartwick, and R. A. Kandel p53 Alterations and Protein Accumulation in Benign Breast Tissue and Breast Cancer Risk: A Cohort Study. Cancer Epidemiol. Biomarkers Prev., July 1, 2006; 15(7): 1316 - 1323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Mitra, C Misra, R K Singh, C K Panda, and S Roychoudhury Association of specific genotype and haplotype of p53 gene with cervical cancer in India J. Clin. Pathol., January 1, 2005; 58(1): 26 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Listgarten, S. Damaraju, B. Poulin, L. Cook, J. Dufour, A. Driga, J. Mackey, D. Wishart, R. Greiner, and B. Zanke Predictive Models for Breast Cancer Susceptibility from Multiple Single Nucleotide Polymorphisms Clin. Cancer Res., April 15, 2004; 10(8): 2725 - 2737. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A-M Martin, P A Kanetsky, B Amirimani, T A Colligon, G Athanasiadis, H A Shih, M R Gerrero, K Calzone, T R Rebbeck, and B L Weber Germline TP53 mutations in breast cancer families with multiple primary cancers: is TP53 a modifier of BRCA1? J. Med. Genet., April 1, 2003; 40(4): e34 - 34. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Leroy, C. Haioun, E. Lepage, N. Le Metayer, F. Berger, E. Labouyrie, V. Meignin, B. Petit, C. Bastard, G. Salles, et al. p53 gene mutations are associated with poor survival in low and low-intermediate risk diffuse large B-cell lymphomas Ann. Onc., July 1, 2002; 13(7): 1108 - 1115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. K. MacLachlan, R. Takimoto, and W. S. El-Deiry BRCA1 Directs a Selective p53-Dependent Transcriptional Response towards Growth Arrest and DNA Repair Targets Mol. Cell. Biol., June 15, 2002; 22(12): 4280 - 4292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Wu, H. Zhao, C. I. Amos, S. Shete, N. Makan, W. K. Hong, F. F. Kadlubar, and M. R. Spitz p53 Genotypes and Haplotypes Associated With Lung Cancer Susceptibility and Ethnicity J Natl Cancer Inst, May 1, 2002; 94(9): 681 - 690. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. D. Hirschi, J. A. Kreps, and K. K. Hirschi Molecular Approaches to Studying Nutrient Metabolism and Function: An Array of Possibilities J. Nutr., May 1, 2001; 131(5): 1605S - 1609. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
), VDS (
), BT-16 (
), and BT-102 (
)
cells was determined 48 h following treatment with varying doses
of cisplatinum (5, 10, or 20 µg/ml) using trypan blue dye exclusion
and confirmed with FACS analysis. The data shown are representative of
mean ± SE of three independent experiments.
B, p53 wild-type cells (VDS) and the
p53 13964GC mutant cell lines (BT-16 and
BT-102) were plated and allowed to attach before being exposed to 10
µg/ml cisplatinum (gray columns) or PBS (white
columns). The p53 cells that underwent transient transfection,
BT-16/p53T and BT-102/p53T, were also treated with 10 µg/ml
cisplatinum (black columns). After 48 h,
quantification of apoptosis was conducted by two independent observers
on at least 300 cells/sample. The apoptotic index is the percentage of
cells with morphological evidence of apoptosis as determined by
staining of nuclei with bis-benzimide Hoechst stain. Results presented
are mean ± SE of three independent experiments (mean
values shown above columns).
