Development of an in Vitro Model to Study Carcinogen-induced Neoplastic Progression of Initiated Mouse Epidermal Cells

David Morgan, David Welty, Adam Glick, David Greenhalgh, Henry Hennings, Stuart H. Yuspa

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Initiation and promotion in mouse skin carcinogenesis produce multiple benign tumors, squamous papillomas, but only a few squamous cell carcinomas. The spontaneous conversion from the benign to the malignant phenotype occurs over many months and in stages, but induced malignant conversion can be accomplished more rapidly by exposure of papilloma-bearing mice to mutagens or by transfection of papilloma cell lines with specific oncogenes. The analysis of genetic targets responsible for carcinogen-induced neoplastic progression would be facilitated by the development of in vitro models where the process is rapid, focal, and quantitative. To this end, primary newborn mouse keratinocytes were initiated in vitro by the introduction of the v-rasHa oncogene via a defective retrovirus. Recipient cells produce squamous papillomas and have a high proliferation rate in culture medium with 0.05 mM Ca2+, but fail to grow in medium with 0.5 mM Ca2+ which is permissive for growth of malignant keratinocytes. When v-rasHa-keratinocytes were exposed to mutagens in vitro, proliferative foci emerged after culture in 0.5 mM Ca2+ for 4 weeks. These foci stained intensely red with rhodamine stain, could be easily quantitated, and readily incorporated bromodeoxyuridine. Dose-response studies with several mutagens indicated that the number of foci increased with concentration to the point where excessive cytotoxicity developed. Mutagens varied in potency for producing foci in the following order: cis-diamminedichloroplatinum > benzo(a)pyrene diolexpoxide I > Nmethyl-N'-nitro-N-nitrosoguanidine > 4-nitroquinoline-N-oxide > N-acetoxy-acetyl-aminofluorene. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate was inactive in the assay. A subset of cell lines derived from foci produced malignant tumors in vivo, while others were not tumorigenic. Analysis of DNA from cell lines and tumors revealed that most tumorigenic cell lines maintained the v-ros”* genome, whereas the viral sequences were deleted in nontumorigenic cell lines. Immunohistochemical analysis indicated that proliferative foci and quiescent v-rasHa keratinocytes expressed keratin 8, a marker of v-rasHa expression in cultured keratinocytes. Cells in foci, but not v-rasHa control cells, expressed keratin 13, a marker which is strongly associated with the malignant progression of skin tumors in vivo. This in vitro assay provides a quantitative model to study chemically induced focal neoplastic progression at the cellular level and to identify agents which may be selective for enhancing malignant conversion.

Original languageEnglish (US)
Pages (from-to)3145-3156
Number of pages12
JournalCancer Research
Volume52
Issue number11
StatePublished - Jun 1992

Fingerprint

Keratinocytes
Carcinogens
Mutagens
Papilloma
Cell Line
Oncogenes
Keratin-13
4-Nitroquinoline-1-oxide
Keratin-8
Nitrosoguanidines
Neoplasms
Skin
Rhodamines
Viral Genome
Benzo(a)pyrene
Tetradecanoylphorbol Acetate
Retroviridae
Bromodeoxyuridine
Tumor Cell Line
Cisplatin

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

Cite this

Morgan, D., Welty, D., Glick, A., Greenhalgh, D., Hennings, H., & Yuspa, S. H. (1992). Development of an in Vitro Model to Study Carcinogen-induced Neoplastic Progression of Initiated Mouse Epidermal Cells. Cancer Research, 52(11), 3145-3156.
Morgan, David ; Welty, David ; Glick, Adam ; Greenhalgh, David ; Hennings, Henry ; Yuspa, Stuart H. / Development of an in Vitro Model to Study Carcinogen-induced Neoplastic Progression of Initiated Mouse Epidermal Cells. In: Cancer Research. 1992 ; Vol. 52, No. 11. pp. 3145-3156.
@article{977524bace8b49d3bbced38bae6b1238,
title = "Development of an in Vitro Model to Study Carcinogen-induced Neoplastic Progression of Initiated Mouse Epidermal Cells",
abstract = "Initiation and promotion in mouse skin carcinogenesis produce multiple benign tumors, squamous papillomas, but only a few squamous cell carcinomas. The spontaneous conversion from the benign to the malignant phenotype occurs over many months and in stages, but induced malignant conversion can be accomplished more rapidly by exposure of papilloma-bearing mice to mutagens or by transfection of papilloma cell lines with specific oncogenes. The analysis of genetic targets responsible for carcinogen-induced neoplastic progression would be facilitated by the development of in vitro models where the process is rapid, focal, and quantitative. To this end, primary newborn mouse keratinocytes were initiated in vitro by the introduction of the v-rasHa oncogene via a defective retrovirus. Recipient cells produce squamous papillomas and have a high proliferation rate in culture medium with 0.05 mM Ca2+, but fail to grow in medium with 0.5 mM Ca2+ which is permissive for growth of malignant keratinocytes. When v-rasHa-keratinocytes were exposed to mutagens in vitro, proliferative foci emerged after culture in 0.5 mM Ca2+ for 4 weeks. These foci stained intensely red with rhodamine stain, could be easily quantitated, and readily incorporated bromodeoxyuridine. Dose-response studies with several mutagens indicated that the number of foci increased with concentration to the point where excessive cytotoxicity developed. Mutagens varied in potency for producing foci in the following order: cis-diamminedichloroplatinum > benzo(a)pyrene diolexpoxide I > Nmethyl-N'-nitro-N-nitrosoguanidine > 4-nitroquinoline-N-oxide > N-acetoxy-acetyl-aminofluorene. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate was inactive in the assay. A subset of cell lines derived from foci produced malignant tumors in vivo, while others were not tumorigenic. Analysis of DNA from cell lines and tumors revealed that most tumorigenic cell lines maintained the v-ros”* genome, whereas the viral sequences were deleted in nontumorigenic cell lines. Immunohistochemical analysis indicated that proliferative foci and quiescent v-rasHa keratinocytes expressed keratin 8, a marker of v-rasHa expression in cultured keratinocytes. Cells in foci, but not v-rasHa control cells, expressed keratin 13, a marker which is strongly associated with the malignant progression of skin tumors in vivo. This in vitro assay provides a quantitative model to study chemically induced focal neoplastic progression at the cellular level and to identify agents which may be selective for enhancing malignant conversion.",
author = "David Morgan and David Welty and Adam Glick and David Greenhalgh and Henry Hennings and Yuspa, {Stuart H.}",
year = "1992",
month = "6",
language = "English (US)",
volume = "52",
pages = "3145--3156",
journal = "Cancer Research",
issn = "0008-5472",
number = "11",

}

Morgan, D, Welty, D, Glick, A, Greenhalgh, D, Hennings, H & Yuspa, SH 1992, 'Development of an in Vitro Model to Study Carcinogen-induced Neoplastic Progression of Initiated Mouse Epidermal Cells', Cancer Research, vol. 52, no. 11, pp. 3145-3156.

Development of an in Vitro Model to Study Carcinogen-induced Neoplastic Progression of Initiated Mouse Epidermal Cells. / Morgan, David; Welty, David; Glick, Adam; Greenhalgh, David; Hennings, Henry; Yuspa, Stuart H.

In: Cancer Research, Vol. 52, No. 11, 06.1992, p. 3145-3156.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Development of an in Vitro Model to Study Carcinogen-induced Neoplastic Progression of Initiated Mouse Epidermal Cells

AU - Morgan, David

AU - Welty, David

AU - Glick, Adam

AU - Greenhalgh, David

AU - Hennings, Henry

AU - Yuspa, Stuart H.

PY - 1992/6

Y1 - 1992/6

N2 - Initiation and promotion in mouse skin carcinogenesis produce multiple benign tumors, squamous papillomas, but only a few squamous cell carcinomas. The spontaneous conversion from the benign to the malignant phenotype occurs over many months and in stages, but induced malignant conversion can be accomplished more rapidly by exposure of papilloma-bearing mice to mutagens or by transfection of papilloma cell lines with specific oncogenes. The analysis of genetic targets responsible for carcinogen-induced neoplastic progression would be facilitated by the development of in vitro models where the process is rapid, focal, and quantitative. To this end, primary newborn mouse keratinocytes were initiated in vitro by the introduction of the v-rasHa oncogene via a defective retrovirus. Recipient cells produce squamous papillomas and have a high proliferation rate in culture medium with 0.05 mM Ca2+, but fail to grow in medium with 0.5 mM Ca2+ which is permissive for growth of malignant keratinocytes. When v-rasHa-keratinocytes were exposed to mutagens in vitro, proliferative foci emerged after culture in 0.5 mM Ca2+ for 4 weeks. These foci stained intensely red with rhodamine stain, could be easily quantitated, and readily incorporated bromodeoxyuridine. Dose-response studies with several mutagens indicated that the number of foci increased with concentration to the point where excessive cytotoxicity developed. Mutagens varied in potency for producing foci in the following order: cis-diamminedichloroplatinum > benzo(a)pyrene diolexpoxide I > Nmethyl-N'-nitro-N-nitrosoguanidine > 4-nitroquinoline-N-oxide > N-acetoxy-acetyl-aminofluorene. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate was inactive in the assay. A subset of cell lines derived from foci produced malignant tumors in vivo, while others were not tumorigenic. Analysis of DNA from cell lines and tumors revealed that most tumorigenic cell lines maintained the v-ros”* genome, whereas the viral sequences were deleted in nontumorigenic cell lines. Immunohistochemical analysis indicated that proliferative foci and quiescent v-rasHa keratinocytes expressed keratin 8, a marker of v-rasHa expression in cultured keratinocytes. Cells in foci, but not v-rasHa control cells, expressed keratin 13, a marker which is strongly associated with the malignant progression of skin tumors in vivo. This in vitro assay provides a quantitative model to study chemically induced focal neoplastic progression at the cellular level and to identify agents which may be selective for enhancing malignant conversion.

AB - Initiation and promotion in mouse skin carcinogenesis produce multiple benign tumors, squamous papillomas, but only a few squamous cell carcinomas. The spontaneous conversion from the benign to the malignant phenotype occurs over many months and in stages, but induced malignant conversion can be accomplished more rapidly by exposure of papilloma-bearing mice to mutagens or by transfection of papilloma cell lines with specific oncogenes. The analysis of genetic targets responsible for carcinogen-induced neoplastic progression would be facilitated by the development of in vitro models where the process is rapid, focal, and quantitative. To this end, primary newborn mouse keratinocytes were initiated in vitro by the introduction of the v-rasHa oncogene via a defective retrovirus. Recipient cells produce squamous papillomas and have a high proliferation rate in culture medium with 0.05 mM Ca2+, but fail to grow in medium with 0.5 mM Ca2+ which is permissive for growth of malignant keratinocytes. When v-rasHa-keratinocytes were exposed to mutagens in vitro, proliferative foci emerged after culture in 0.5 mM Ca2+ for 4 weeks. These foci stained intensely red with rhodamine stain, could be easily quantitated, and readily incorporated bromodeoxyuridine. Dose-response studies with several mutagens indicated that the number of foci increased with concentration to the point where excessive cytotoxicity developed. Mutagens varied in potency for producing foci in the following order: cis-diamminedichloroplatinum > benzo(a)pyrene diolexpoxide I > Nmethyl-N'-nitro-N-nitrosoguanidine > 4-nitroquinoline-N-oxide > N-acetoxy-acetyl-aminofluorene. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate was inactive in the assay. A subset of cell lines derived from foci produced malignant tumors in vivo, while others were not tumorigenic. Analysis of DNA from cell lines and tumors revealed that most tumorigenic cell lines maintained the v-ros”* genome, whereas the viral sequences were deleted in nontumorigenic cell lines. Immunohistochemical analysis indicated that proliferative foci and quiescent v-rasHa keratinocytes expressed keratin 8, a marker of v-rasHa expression in cultured keratinocytes. Cells in foci, but not v-rasHa control cells, expressed keratin 13, a marker which is strongly associated with the malignant progression of skin tumors in vivo. This in vitro assay provides a quantitative model to study chemically induced focal neoplastic progression at the cellular level and to identify agents which may be selective for enhancing malignant conversion.

UR - http://www.scopus.com/inward/record.url?scp=0026716304&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026716304&partnerID=8YFLogxK

M3 - Article

C2 - 1375535

AN - SCOPUS:0026716304

VL - 52

SP - 3145

EP - 3156

JO - Cancer Research

JF - Cancer Research

SN - 0008-5472

IS - 11

ER -