The ras Superfamily of GTPases (1993)

Juan Carlos Lacal, Frank Patrick McCormick

• Publisher: CRC Press
• Year: 2017
• ISBN:
  9781351356848
• Edition: 1
• Topic: Ciencias biológicas
• Subtopic: Biología celular

The ras Superfamily of GTPases presents the most comprehensive compilation of information available regarding aspects of the putative function of small ras-related GTPases. The book's chapters were written by the world's most prominent scientists in this field and cover such topics as the structure and properties of ras proteins, ras function, the ras superfamily in general, and the functional regulation of ras and ras-related GTPases. The book will benefit cell biologists, oncologists, neurobiologists, molecular biologists, and others interested in the topic.

SECTION I
The World of ras Genes and Their Products

• Oncogenic Potential of ras Genes

• Structure and Properties of ras Proteins

• Genetic Approaches to ras Function

Chapter 1

ras GENES INVOLVEMENT IN CARCINOGENESIS: LESSONS FROM ANIMAL MODEL SYSTEMS

Javier León And Angel Pellicer

TABLE OF CONTENTS

I. Introduction and Historical Perspective

II. Animal Model Systems and ras Activation

A. Detection of ras-Mutated Genes

1. Biological Assays

2. Molecular Assays

a. Gene Cloning

b. RNase Mismatch Cleavage Analysis

c. Oligonucleotide Differential Hybridization in Gel

d. Allele-Specific Oligonucleotide Hybridization of PCR Products

e. Restriction Fragment Length Polymorphism (RFLP)

f. Direct Sequencing of Amplified DNA

B. Skin Tumors

C. Mammary Tumors

D. Lymphomas

E. Liver Tumors

F. Lung Tumors and Other Induced Tumors

III. ras Activation and Tissue Specificity

IV. Carcinogen-Specific Mutation of ras Genes

V. Is ras Mutation an Early or Late Event in Tumorigenesis?

A. Evidence in Favor of ras Gene Mutation as a Late Event

B. Evidence in Favor of ras Activation as an Early Event

VI. ras Activation is not Sufficient for Carcinogenesis

A. Lessons from Carcinogenesis Model Systems

B. Lessons from Transgenic Animals

1. Activated ras Genes Are Not Sufficient to Induce a Frank Malignant Tumor

2. There Exists a Tissue Specificity for the Effects of ras Transgenes Not Dependent on the Promoter

3. Other Oncogenes Cooperate with ras in Transgenic Tumor Development

VII. Final Remarks

Acknowledgments

References

I. Introduction and Historical Perspective

ras oncogenes were originally discovered as part of the genomes of highly oncogenic murine retroviruses. At least five sarcoma viruses containing activated versions of ras genes have been isolated (reviewed by Lacal and Tronick¹). All these tumors cause sarcomas in rats or mice, the Harvey sarcoma virus and the Kirsten sarcoma virus being the representative isolates carrying the activated forms of H- and K-ras genes, respectively. Thus, the story of ras genes and ras-related genes is linked to animal carcinogenesis. Years later (1982) several groups showed that genes isolated from human tumor cell lines by transfection methods were homologs to the oncogene from Harvey sarcoma virus. This gene was a mutated version from the normal H-ras found in nontumor cells.^{2, 3, 4}, and ⁵ Since then, activated ras genes have been found in numerous human and animal tumors.

The mammalian ras gene subfamily belongs to the superfamily of small GTPases (reviewed in References 6 and 7 and Section II of this book). The ras subfamily has three members: H-, K-, and N-ras. ras proteins (termed p21 for their molecular mass, 21 kDa) contain 188 (K-ras-B) or 189 (H- N-, and K-ras-A) amino acids. The first 80 amino acids are almost completely conserved among the four proteins. The protein segment encompassing amino acids 80 to 164 show slightly less similarity among ras proteins (70 to 80%). The rest of the protein, the hypervariable region, is specific for each ras gene, except for the last four amino acids.^8-9

ras protooncogenes, as well as neu, are activated by point mutations in their coding sequences, although overexpression of raj genes can also transform cells in vitro.^{10, 11, 12, 13} and ¹⁴ Activated ras oncogenes contain point mutations affecting either codons 12 (Gly), 13 (Gly), or 61 (Gin). Rarely, mutations on other codons (e.g., in codon 117¹⁵ or 146¹⁶) have also been reported.

The fact that ras genes are activated by point mutations opens the way for the search of the molecular mechanisms relating the genetic injury by the carcinogenic agent with the uncontrolled cell proliferation leading to tumor formation.

II. ANIMAL MODEL SYSTEMS AND ras ACTIVATION

A large body of data has been published over the last decade on ras activation in human tumors, and significant ras activation frequencies have been linked to several types of human tumors. Despite that, few general conclusions can be drawn on the involvement of ras genes in tumor generation based on human studies. In human carcinogenesis, key parameters as to the carcinogenic agent and genetic background are frequently unknown. Therefore, a considerable number of animal model systems have been established to study carcinogenesis at the molecular level. Some model systems are based on selected animal strains showing high frequencies of certain spontaneous tumors. Nevertheless, most models are based on protocols that use a wide variety of carcinogenic agents to treat particular animal species or strains with increased sensitivity to the carcinogen.

Although a large variety of model systems have been designed for carcin-ogenetic studies, only a few have been analyzed for oncogene detection. Previous reviews^{17, 18,} and ¹⁹ dealt with oncogene activation in animal carcinogenesis. Here we will briefly review the results obtained on ras oncogene activation in animal model systems, albeit the list of models cited is not exhaustive, ras genes carrying point mutations are by far the most prevalent oncogenes found both in human cancer and in experimental carcinogenesis, ras gene amplification or increased expression have occasionally been reported in some animal models,^{20, 21,} and ²² as well as some human tumors (reviewed by Field and Spandidos²³). However, here we will consider ras activation as synonymous with point mutation. Apart from ras activation, few activated oncogenes have been consistently found in tumors of animal model systems. These oncogenes include mutated neu genes²⁴ and rearranged or amplified c-myc.^{25, 26} Detection of other oncogenes has rarely been reported. The prevalence of ras oncogenes found in experimental carcinogenesis correlates with the situation in human cancer, where activated ras genes are again the oncogenes most commonly found (reviewed by Bos²⁷). Although there are some correlations between the finding of particular ras genes in human and animal induced tumors (i.e., squamous cell carcinomas, keratoacanthomas, and bladder and lung carcinomas) this is not the general rule.

The high frequency of ras activation could be partly explained because the methodology originally used (NIH 3T3 transformation assays, see below) was biased in favor of ras oncogene detection. As a consequence, mutations in ras genes are more actively sought than alterations on other genes. However, as discussed below, the high prevalence of mutated ras genes in induced and spontaneous tumors speaks in favor of important roles for these genes in the cellular proliferation and differentiation control systems.

A. DETECTION OF ras-MUTATED GENES

In model systems, ras activation has been detected by one or more of the following methods.

1. Biological Assays

ras-activated genes introduced in rodent fibroblast cell lines, such as mouse NIH 3T3, induce cellular transformation in a dominant fashion. Therefore, activated ras oncogenes can be detected either by a focus-forming assay in vitro or by tumor formation in nude mice after injection of the transfected cells. As noted above, the first assay led to the discovery of cellular ras oncogenes. However, the focus-forming assay is frequently replaced by the more sensitive nude mice assay.^28-29

2. Molecular Assays

a. Gene Cloning

After the focus-forming assay mentioned above, the first method used in ras oncogene detection, was the cloning and sequencing of the genes out of the transformed cells in order to identify the activating mutation of the ras gene. Today, this identification can be more easily done by the application of ...

Table of contents

• Cover Page
• Half title
• Title Page
• Copyright Page
• Preface
• The Editors
• Contributors
• Table of Contents
• I. The World of ras Genes and Their Products
• II. The ras Superfamily
• III. Functional Regulation of ras and ras-Related GTPases
• Index