Oncogenes in Laryngeal Cancer: Serial Passage of Transformed Cellular DNA

1985 ◽  
Vol 93 (3) ◽  
pp. 346-350 ◽  
Author(s):  
William H. Friedman ◽  
Barry N. Rosenblum ◽  
Paul Loewenstein ◽  
Helen Thornton ◽  
George Katsantonis ◽  
...  
Keyword(s):  
Laryngeal Cancer ◽  
Dna Sequences ◽  
Squamous Cell Cancer ◽  
Cell Cancer ◽  
Serial Passage ◽  
3T3 Cells ◽  
Human Dna ◽  
Cellular Dna ◽  
Nih 3T3 ◽  
Narrow Bands

DNA originally extracted from squamous cell cancer of the larynx has been serially passaged through transformed populations of NIH/3T3 mouse fibroblasts. The transformed foci were then harvested, cloned to volume, and incubated with a fresh population of NIH/3T3 cells in a second passage. Transforming efficiencies were enhanced by serial passage. In addition, Southern Blot analysis of the transformed foci revealed hybridization between transformant DNA and human probe DNA from the Alu family of conserved human DNA sequences. In the first passage this hybridization took the form of diffuse homology throughout the entire molecular weight distribution. The second-passage DNA showed “narrow bands” indicating the possibility that an oncogene has been identified in laryngeal cancer and that serial passage has eliminated contaminating human sequences. Repetitive transfection in third- and fourth-passage studies is now being completed.

scholarly journals Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

1983 ◽  
Vol 3 (7) ◽  
pp. 1182-1186 ◽  
Author(s):  
Nancy H. Colburn ◽  
Catherine B. Talmadge ◽  
Thomas D. Gindhart
Keyword(s):  
Phorbol Ester ◽  
Dna Sequences ◽  
Genetic Basis ◽  
Tumor Promoters ◽  
3T3 Cells ◽  
Dominant Trait ◽  
Jb6 Cells ◽  
Cellular Dna ◽  
Common Parent ◽  
Nih 3T3

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P+) and nonpromotable (P−) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P+lines, JB6 Cl 41 or JB6 Cl 22, was CaPO4precipitated and used to transfect the P−cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 × 10−8M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P−JB6 Cl 30 cells yielded 40 to 50 colonies per 105cells. In contrast, transfection of Cl 30 cells with “P+DNA” derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 105cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P+DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P+lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P+DNA, no evidence for acquisition of promotability was obtained. P−JB6 Cl 25, like Cl 30, also permitted expression of transfected P+DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P+DNA and requires recipient cells of the appropriate phenotype for expression.

Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

1983 ◽  
Vol 3 (7) ◽  
pp. 1182-1186
Author(s):  
Nancy H. Colburn ◽  
Catherine B. Talmadge ◽  
Thomas D. Gindhart
Keyword(s):  
Phorbol Ester ◽  
Dna Sequences ◽  
Genetic Basis ◽  
Tumor Promoters ◽  
3T3 Cells ◽  
Dominant Trait ◽  
Jb6 Cells ◽  
Cellular Dna ◽  
Common Parent ◽  
Nih 3T3

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P + ) and nonpromotable (P − ) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12- o -tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P + lines, JB6 Cl 41 or JB6 Cl 22, was CaPO 4 precipitated and used to transfect the P − cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 × 10 −8 M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P − JB6 Cl 30 cells yielded 40 to 50 colonies per 10 5 cells. In contrast, transfection of Cl 30 cells with “P + DNA” derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10 5 cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P + DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P + lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P + DNA, no evidence for acquisition of promotability was obtained. P − JB6 Cl 25, like Cl 30, also permitted expression of transfected P + DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P + DNA and requires recipient cells of the appropriate phenotype for expression.

Molecular cloning and characterization of an activated human c-raf-1 gene

1987 ◽  
Vol 7 (5) ◽  
pp. 1776-1781
Author(s):  
M Fukui ◽  
T Yamamoto ◽  
S Kawai ◽  
F Mitsunobu ◽  
K Toyoshima
Keyword(s):  
Dna Sequences ◽  
Promoter Sequence ◽  
Small Population ◽  
Dna Transfection ◽  
Amino Terminal ◽  
Human Dna ◽  
Nih 3T3 ◽  
Amino Terminal Sequence ◽  
Tumor Dna

Results of previous studies have shown that a raf-related transforming DNA sequence is present in NIH 3T3 transformants that are derived from GL-5-JCK human glioblastoma DNA transfection. The transforming DNA was molecularly cloned by using cosmid vector pJB8 to determine its structure and origin. Analyses of selected clones revealed that the transforming DNA consisted of three portions of human DNA sequences, with the 3' half of the c-raf-1 gene as its middle portion. This raf region was about 20 kilobases long and contained exons 8 to 17 and the poly(A) addition site. RNA blot analysis showed that the raf-related transforming DNA was transcribed into 5.3-, 4.8-, and 2.5-kilobase mRNAs; the 2.5-kilobase transcript was thought to be the major transcript. Immunoprecipitation analyses revealed that a 44-kilodalton raf-related protein was specifically expressed in the NIH 3T3 transformants. The raf-related transforming DNA was considered to be activated when its amino-terminal sequence was truncated and the DNA was coupled with a foreign promoter sequence. On hybridization analysis of the original GL-5-JCK glioblastoma DNA, no rearrangement of c-raf-1 was detectable in the tumor DNA. The rearrangement of c-raf-1 may have occurred during transfection or may have been present in a small population of the original tumor cells as a result of tumor progression.

scholarly journals NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

1985 ◽  
Vol 5 (1) ◽  
pp. 259-262 ◽  
Author(s):  
U P Thorgeirsson ◽  
T Turpeenniemi-Hujanen ◽  
J E Williams ◽  
E H Westin ◽  
C A Heilman ◽  
...  
Keyword(s):  
Nude Mice ◽  
Dna Sequences ◽  
Immune Cell ◽  
Human Tumor ◽  
3T3 Cells ◽  
Metastatic Phenotype ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Tumor Dna ◽  
Nih 3T3 Cells

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.

scholarly journals Transfected human beta-polymerase promoter contains a ras-responsive element.

1990 ◽  
Vol 10 (7) ◽  
pp. 3852-3856 ◽  
Author(s):  
P S Kedar ◽  
D R Lowy ◽  
S G Widen ◽  
S H Wilson
Keyword(s):  
Dna Repair ◽  
Transient Expression ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
3T3 Cells ◽  
Gap Filling ◽  
Transient Expression Assay ◽  
Responsive Element ◽  
Cellular Dna ◽  
Nih 3T3

beta-Polymerase is a vertebrate cellular DNA polymerase involved in gap-filling synthesis during some types of genomic DNA repair. We report that a cloned human beta-polymerase promoter in a transient expression assay is activated by p21v-rasH expression in NIH 3T3 cells. A decanucleotide palindromic element, GTGACGTCAC, at positions -49 to -40 in the promoter is required for this ras-mediated stimulation.

Comparison of DNA Content Parameters in Paired, Fresh Tissue Pretreatment Biopsies and Surgical Resections from Squamous Cell Carcinoma of the Head and Neck

2003 ◽  
Vol 128 (2) ◽  
pp. 169-177 ◽  
Author(s):  
B. F. EL Rayes ◽  
Z. Maciorowski ◽  
H. Pietraszkiewicz ◽  
J. F. Ensley
Keyword(s):  
Head And Neck ◽  
Squamous Cell ◽  
Dna Ploidy ◽  
Squamous Cell Cancer ◽  
Cell Cancer ◽  
Poor Quality ◽  
Fresh Tissue ◽  
Cellular Dna ◽  
Neck Squamous Cell Cancer ◽  
Dna Characteristics

OBJECTIVE: Cellular DNA characteristics derived from pretreatment biopsy (PTB) may become important for predicting treatment outcomes in patients with head and neck squamous cell cancer (HNSCC). Whether the PTB adequately represents the whole specimen is of critical importance. STUDY DESIGN: In a series of >700 HNSCCs, we identified 59 cases in which the PTB and the surgical resection (SR) met the following criteria: PTB and SR were from the same site, and SR was obtained within 5 weeks of PTB with no intervening treatments. RESULTS: Twenty-nine percent of the PTB specimens were DNA diploid. Only 1 of the 11 subsequent DNA diploid SR was associated with a DNA aneuploid PTB (91% concordance). Of the 48 DNA aneuploid tumors, 3 were associated with DNA diploid PTB (94% concordance). Three other DNA aneuploid SRs were associated with PTB of poor quality. CONCLUSION: With respect to DNA ploidy, PTB are representative of SR specimens.

scholarly journals Identification of ErbB3-stimulated genes using modified representational difference analysis

1997 ◽  
Vol 323 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Carl F. EDMAN ◽  
Sally A. PRIGENT ◽  
Andrea SCHIPPER ◽  
James R. FERAMISCO
Keyword(s):  
Dna Sequences ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Representational Difference Analysis ◽  
3T3 Cells ◽  
Difference Analysis ◽  
Total Rna ◽  
Egfr Family ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The epidermal growth factor receptor (EGFR) family of tyrosine kinases is involved in the growth of normal and tumour cells. The specific contribution of each of the four family members to these processes remains unclear. In the present study we have used a PCR-based subtractive approach to identify differences in messages induced in response to activation of ErbB3 and EGFR. The approach described is a modification of the representational difference analysis technique adapted for analysis of cDNA, which we have modified to permit identification of differential gene expression using as little as 20 μg of total RNA as the starting material. The mRNA obtained from EGF-stimulated NIH-3T3 cells expressing chimaeric EGFR-ErbB3 receptors provided the tester amplicons (small PCR-amplified fragments) which were subtracted against driver amplicons derived from unstimulated NIH-3T3 cells expressing the EGFR-ErbB3 chimaera or EGF-stimulated NIH-3T3 cells overexpressing the EGFR. A total of 22 different clones were isolated, 90% of which showed increased expression in the tester amplicons. Six of these, corresponding to known DNA sequences, were selected for further Northern blot analysis against total RNA prepared from the starting cell lines. Of these, the gene encoding the protein dlk (or a closely related protein, Pref-1) was identified as being regulated by ErbB3 but not by the EGFR. Other genes appeared to be elevated by both ErbB3 and EGFR, including those encoding c-jun, Ret finger protein (RFP), neuroleukin and amyloid protein precursor. One gene product, TIS11, was identified as being regulated by EGFR but not by ErbB3.

scholarly journals Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells.

1987 ◽  
Vol 7 (7) ◽  
pp. 2512-2520 ◽  
Author(s):  
R D Owen ◽  
M C Ostrowski
Keyword(s):  
Dna Sequences ◽  
Regulation Of Gene Expression ◽  
Hormone Treatment ◽  
Northern Blotting ◽  
3T3 Cells ◽  
Ras Gene ◽  
Cellular Genes ◽  
Conditional Expression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-related RNA and retrovirus-related VL30 RNA. Run-on transcription in isolated nuclei shows that the increase in v-sis-related RNA is not dependent on transcription and therefore must arise by a post-transcriptional mechanism. The increase in VL30 expression is a transcriptional effect. Hormone treatment of normal NIH 3T3 cells has no effect on the expression of these DNA sequences. These results suggest that v-ras stimulation of autocrine factors may play a role in transformation of cells by this gene and also suggest a reverse genetic strategy to determine the nucleic acid sequences and cellular factors involved in the regulation of gene expression that is observed.

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice

1985 ◽  
Vol 5 (1) ◽  
pp. 259-262
Author(s):  
U P Thorgeirsson ◽  
T Turpeenniemi-Hujanen ◽  
J E Williams ◽  
E H Westin ◽  
C A Heilman ◽  
...  
Keyword(s):  
Nude Mice ◽  
Dna Sequences ◽  
Immune Cell ◽  
Human Tumor ◽  
3T3 Cells ◽  
Metastatic Phenotype ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Tumor Dna ◽  
Nih 3T3 Cells

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.

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