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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 for Alu I-digested and undigested DNA from stilbene estrogen-induced kidney tumors (T) and age-matched control (C) tissue. Arrows indicate the RAPD loci harboring mutations in the Alu I restriction enzyme recognition sequence. Note that these mutations were not detected in the RAPDs from undigested DNA using the same primers.
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Figure 5: Representative RAPD fingerprints for Alu I-digested and undigested DNA from stilbene estrogen-induced kidney tumors (T) and age-matched control (C) tissue. Arrows indicate the RAPD loci harboring mutations in the Alu I restriction enzyme recognition sequence. Note that these mutations were not detected in the RAPDs from undigested DNA using the same primers.

Mentions: Of the 44 mutated loci that were amplified, 37 (84.10%) harbored mutations only in tumor DNA and not in the adjacent uninvolved kidney tissue DNA, as determined through comparison with kidney DNA from age-matched control hamsters. Representative fingerprints are shown in Figs. 2,3,4,5. The sizes of 37 individual loci, and the sequence of the primers used to amplify them, are given in Table 2. Of these 37 mutated loci, Fig. 3 shows tumor DNA-specific losses in the 1.55-, 1.0-, 0.54-kbp products amplified with primer OPA 08, the 0.3-kbp fragment amplified with OPK02, the 1.15-kbp fragment amplified with OPK 09, and the 3.1-kb product amplified by OPK 13. Fig. 4 shows that losses of the 1.95-kbp product amplified using primer OPC 13, the 1.6- and 0.7-kbp fragments amplified with primer OPE 03, the 1.6-, and 1.0-kbp products amplified with primer OPK 16, the 1.75-kbp fragment produced with primer OP 26-12, and the 1.2-kbp product amplified with primer OP 26-25 also were specific to reactions conducted with tumor DNA. In addition, loss of the 0.3-kbp product amplified with primer OPC 01 and the 1.1-kb fragment produced with OPC14 was observed in reactions conducted with tumor DNA and not with DNA from uninvolved (non-tumor) surrounding tissue adjacent to tumors (Table 2). The gain of 18 loci (0.75 kbp with OPA 08, 1.2 kbp with OPA 15, 1.9 and 1.35 kbp with OPK 09 [shown in Fig. 3], and 2.0, 1.5, 1.2 and 0.8 kbp with OPE 03 [shown in Fig. 4]) was observed in the tumor DNA as compared to control DNA. Out of the remaining eight loci, seven showed loss of intensity (the 0.55-kbp fragment amplified with primer OPA 08 that is shown in Fig. 3; and the 0.7- and 1.5-kbp products amplified with primer OP 26-07, the 1.6-kbp fragment amplified with primer OP26-12, and 1.25- and 0.8-kbp fragments produced with primer OP 26-25 shown in Fig. 4), and one showed a gain of intensity (the 0.7-kb produced amplified with primer OPA 17) compared to data from age-matched control kidneys.


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

Singh KP, Roy D - J Carcinog (2004)

Representative RAPD fingerprints for Alu I-digested and undigested DNA from stilbene estrogen-induced kidney tumors (T) and age-matched control (C) tissue. Arrows indicate the RAPD loci harboring mutations in the Alu I restriction enzyme recognition sequence. Note that these mutations were not detected in the RAPDs from undigested DNA using the same primers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC398418&req=5

Figure 5: Representative RAPD fingerprints for Alu I-digested and undigested DNA from stilbene estrogen-induced kidney tumors (T) and age-matched control (C) tissue. Arrows indicate the RAPD loci harboring mutations in the Alu I restriction enzyme recognition sequence. Note that these mutations were not detected in the RAPDs from undigested DNA using the same primers.
Mentions: Of the 44 mutated loci that were amplified, 37 (84.10%) harbored mutations only in tumor DNA and not in the adjacent uninvolved kidney tissue DNA, as determined through comparison with kidney DNA from age-matched control hamsters. Representative fingerprints are shown in Figs. 2,3,4,5. The sizes of 37 individual loci, and the sequence of the primers used to amplify them, are given in Table 2. Of these 37 mutated loci, Fig. 3 shows tumor DNA-specific losses in the 1.55-, 1.0-, 0.54-kbp products amplified with primer OPA 08, the 0.3-kbp fragment amplified with OPK02, the 1.15-kbp fragment amplified with OPK 09, and the 3.1-kb product amplified by OPK 13. Fig. 4 shows that losses of the 1.95-kbp product amplified using primer OPC 13, the 1.6- and 0.7-kbp fragments amplified with primer OPE 03, the 1.6-, and 1.0-kbp products amplified with primer OPK 16, the 1.75-kbp fragment produced with primer OP 26-12, and the 1.2-kbp product amplified with primer OP 26-25 also were specific to reactions conducted with tumor DNA. In addition, loss of the 0.3-kbp product amplified with primer OPC 01 and the 1.1-kb fragment produced with OPC14 was observed in reactions conducted with tumor DNA and not with DNA from uninvolved (non-tumor) surrounding tissue adjacent to tumors (Table 2). The gain of 18 loci (0.75 kbp with OPA 08, 1.2 kbp with OPA 15, 1.9 and 1.35 kbp with OPK 09 [shown in Fig. 3], and 2.0, 1.5, 1.2 and 0.8 kbp with OPE 03 [shown in Fig. 4]) was observed in the tumor DNA as compared to control DNA. Out of the remaining eight loci, seven showed loss of intensity (the 0.55-kbp fragment amplified with primer OPA 08 that is shown in Fig. 3; and the 0.7- and 1.5-kbp products amplified with primer OP 26-07, the 1.6-kbp fragment amplified with primer OP26-12, and 1.25- and 0.8-kbp fragments produced with primer OP 26-25 shown in Fig. 4), and one showed a gain of intensity (the 0.7-kb produced amplified with primer OPA 17) compared to data from age-matched control kidneys.

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