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Somatic mutations in stilbene estrogen-induced Syrian hamster kidney tumors identified by DNA fingerprinting.

Singh KP, Roy D - J Carcinog (2004)

Bottom Line: Sixty one of the arbitrary primers amplified 365 loci that contain approximately 476 kbp of the hamster genome.The presence or absence of the amplified DNA fragments indicate homozygous insertions or deletions in the kidney tumor DNA compared to the age-matched normal kidney tissue DNA.Although the exact functional importance of mutated loci is unknown, this study indicates that these altered loci may participate during tumor progression in the kidney.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, AL 35294-0022, USA. royd@uab.edu

ABSTRACT
Kidney tumors from stilbene estrogen (diethylstilbestrol)-treated Syrian hamsters were screened for somatic genetic alterations by Random Amplified Polymorphic DNA-polymerase chain-reaction (RAPD-PCR) fingerprinting. Fingerprints from tumor tissue were generated by single arbitrary primers and compared with fingerprints for normal tissue from the same animal, as well as normal and tumor tissues from different animals. Sixty one of the arbitrary primers amplified 365 loci that contain approximately 476 kbp of the hamster genome. Among these amplified DNA fragments, 44 loci exhibited either qualitative or quantitative differences between the tumor tissues and normal kidney tissues. RAPD-PCR loci showing decreased and increased intensities in tumor tissue DNA relative to control DNA indicate that loci have undergone allelic losses and gains, respectively, in the stilbene estrogen-induced tumor cell genome. The presence or absence of the amplified DNA fragments indicate homozygous insertions or deletions in the kidney tumor DNA compared to the age-matched normal kidney tissue DNA. Seven of 44 mutated loci also were present in the kidney tissues adjacent to tumors (free of macroscopic tumors). The presence of mutated loci in uninvolved (non-tumor) surrounding tissue adjacent to tumors from stilbene estrogen-treated hamsters suggests that these mutations occurred in the early stages of carcinogenesis. The cloning and sequencing of RAPD amplified loci revealed that one mutated locus had significant sequence similarity with the hamster Cyp1A1 gene. The results show the ability of RAPD-PCR to detect and isolate, in a single step, DNA sequences representing genetic alterations in stilbene estrogen-induced cancer cells, including losses of heterozygosity, and homozygous deletion and insertion mutations. RAPD-PCR provides an alternative molecular approach for studying cancer cytogenetics in stilbene estrogen-induced tumors in humans and experimental models. Although the exact functional importance of mutated loci is unknown, this study indicates that these altered loci may participate during tumor progression in the kidney.

No MeSH data available.


Related in: MedlinePlus

Representative RAPD fingerprints showing gains/losses and intensity differences for amplification products indicated by arrow with size of the amplified fragment (kb) common to DNA from both stilbene estrogen-induced kidney tumors (T) and uninvolved surrounding tissue adjacent to tumors, i.e., free of macroscopic tumors (M), in comparison with DNA from age-matched control kidneys (C). Primers used (OPA11, OPA 18, OPA19, OPC 14 and OPE12) are indicated at the bottom of each set of fingerprints.
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Figure 2: Representative RAPD fingerprints showing gains/losses and intensity differences for amplification products indicated by arrow with size of the amplified fragment (kb) common to DNA from both stilbene estrogen-induced kidney tumors (T) and uninvolved surrounding tissue adjacent to tumors, i.e., free of macroscopic tumors (M), in comparison with DNA from age-matched control kidneys (C). Primers used (OPA11, OPA 18, OPA19, OPC 14 and OPE12) are indicated at the bottom of each set of fingerprints.

Mentions: We observed 7 mutated loci amplified by 5 individual primers (OPA11, 18, 19; OPC14, OPE12) in tumor-free tissues that were adjacent to tumors, and these same loci were also altered in the adjacent DES-induced tumors. The sizes of these seven loci are indicated by bold-face type in Table 2, and the RAPD fingerprints are shown in Fig. 2. The mutations consisted of three losses (1.5- and 0.75-kbp fragments with primer OPA11 and a 0.6-kbp fragment with primer OPA19), and four additions (1.8-, 0.71-, 2.0- and 1.15-kb fragments with the OPA 18, OPA 19, OPC 14, and OPE 12 primers, respectively) (Fig. 2). We performed RAPD-PCR analysis on three pools of DNA from kidney tissues of age-matched control animals, each pool containing DNA from two animals. Using the above sets of primers, we did not observe any change in the RAPD-PCR fingerprints between the different batches of untreated controls (data not shown). This indicates that there were no spontaneous mutations at these loci among the control animals, or at least that the frequency was too low to be detected by the analysis. This observation suggests that these mutations were induced as a result of DES treatment and were not spontaneous mutations associated with the aging of animals.


Somatic mutations in stilbene estrogen-induced Syrian hamster kidney tumors identified by DNA fingerprinting.

Singh KP, Roy D - J Carcinog (2004)

Representative RAPD fingerprints showing gains/losses and intensity differences for amplification products indicated by arrow with size of the amplified fragment (kb) common to DNA from both stilbene estrogen-induced kidney tumors (T) and uninvolved surrounding tissue adjacent to tumors, i.e., free of macroscopic tumors (M), in comparison with DNA from age-matched control kidneys (C). Primers used (OPA11, OPA 18, OPA19, OPC 14 and OPE12) are indicated at the bottom of each set of fingerprints.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC398418&req=5

Figure 2: Representative RAPD fingerprints showing gains/losses and intensity differences for amplification products indicated by arrow with size of the amplified fragment (kb) common to DNA from both stilbene estrogen-induced kidney tumors (T) and uninvolved surrounding tissue adjacent to tumors, i.e., free of macroscopic tumors (M), in comparison with DNA from age-matched control kidneys (C). Primers used (OPA11, OPA 18, OPA19, OPC 14 and OPE12) are indicated at the bottom of each set of fingerprints.
Mentions: We observed 7 mutated loci amplified by 5 individual primers (OPA11, 18, 19; OPC14, OPE12) in tumor-free tissues that were adjacent to tumors, and these same loci were also altered in the adjacent DES-induced tumors. The sizes of these seven loci are indicated by bold-face type in Table 2, and the RAPD fingerprints are shown in Fig. 2. The mutations consisted of three losses (1.5- and 0.75-kbp fragments with primer OPA11 and a 0.6-kbp fragment with primer OPA19), and four additions (1.8-, 0.71-, 2.0- and 1.15-kb fragments with the OPA 18, OPA 19, OPC 14, and OPE 12 primers, respectively) (Fig. 2). We performed RAPD-PCR analysis on three pools of DNA from kidney tissues of age-matched control animals, each pool containing DNA from two animals. Using the above sets of primers, we did not observe any change in the RAPD-PCR fingerprints between the different batches of untreated controls (data not shown). This indicates that there were no spontaneous mutations at these loci among the control animals, or at least that the frequency was too low to be detected by the analysis. This observation suggests that these mutations were induced as a result of DES treatment and were not spontaneous mutations associated with the aging of animals.

Bottom Line: Sixty one of the arbitrary primers amplified 365 loci that contain approximately 476 kbp of the hamster genome.The presence or absence of the amplified DNA fragments indicate homozygous insertions or deletions in the kidney tumor DNA compared to the age-matched normal kidney tissue DNA.Although the exact functional importance of mutated loci is unknown, this study indicates that these altered loci may participate during tumor progression in the kidney.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, AL 35294-0022, USA. royd@uab.edu

ABSTRACT
Kidney tumors from stilbene estrogen (diethylstilbestrol)-treated Syrian hamsters were screened for somatic genetic alterations by Random Amplified Polymorphic DNA-polymerase chain-reaction (RAPD-PCR) fingerprinting. Fingerprints from tumor tissue were generated by single arbitrary primers and compared with fingerprints for normal tissue from the same animal, as well as normal and tumor tissues from different animals. Sixty one of the arbitrary primers amplified 365 loci that contain approximately 476 kbp of the hamster genome. Among these amplified DNA fragments, 44 loci exhibited either qualitative or quantitative differences between the tumor tissues and normal kidney tissues. RAPD-PCR loci showing decreased and increased intensities in tumor tissue DNA relative to control DNA indicate that loci have undergone allelic losses and gains, respectively, in the stilbene estrogen-induced tumor cell genome. The presence or absence of the amplified DNA fragments indicate homozygous insertions or deletions in the kidney tumor DNA compared to the age-matched normal kidney tissue DNA. Seven of 44 mutated loci also were present in the kidney tissues adjacent to tumors (free of macroscopic tumors). The presence of mutated loci in uninvolved (non-tumor) surrounding tissue adjacent to tumors from stilbene estrogen-treated hamsters suggests that these mutations occurred in the early stages of carcinogenesis. The cloning and sequencing of RAPD amplified loci revealed that one mutated locus had significant sequence similarity with the hamster Cyp1A1 gene. The results show the ability of RAPD-PCR to detect and isolate, in a single step, DNA sequences representing genetic alterations in stilbene estrogen-induced cancer cells, including losses of heterozygosity, and homozygous deletion and insertion mutations. RAPD-PCR provides an alternative molecular approach for studying cancer cytogenetics in stilbene estrogen-induced tumors in humans and experimental models. Although the exact functional importance of mutated loci is unknown, this study indicates that these altered loci may participate during tumor progression in the kidney.

No MeSH data available.


Related in: MedlinePlus