Transformation of Human Epithelial Cells (1992)

Molecular and Oncogenetic Mechanisms

George Milo, Bruce Casto, Charles Shuler

• Publisher: CRC Press
• Year: 2017
• ISBN:
  9781351355971
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Cell Biology
• Index: Biological Sciences

This book offers a conceptual explanation of the interrelationships that exist between the stages in the progression of initiated epithelial cells in culture compared with the diverse tissue of organs and the progression of tumors from different organ sites. The fate of the modification of adducts is discussed at the molecular level. The role that modifications in hot spots in oncogenes and supressor genes play at the molecular level and how these molecular modifications can lead to an explanation of molecular control in the formation of tumor phenotypes is also examined. Researchers in cell biology and toxicology, applied pharmacology, carcinogenesis, teratogenesis, mutagenesis, and molecular toxicology will find the book useful, interesting reading.

Chapter 1

In Vitro CELLULAR AGING AND IMMORTALIZATION

James R. Smith

TABLE OF CONTENTS

I. Introduction

II. In Vitro Cellular Aging is Dominant in Somatic Cell Hybrids

A. Limited In Vitro Life-Span of Normal Cells

B. Hybrids between Normal and Immortal Cells

C. Fusion of Immortal Cells with Immortal Cell Lines

D. Microcell Hybrid Experiments

III. Cellular Aging Is an Active Process

A. Heterokaryon Experiments

B. Membrane-Associated DNA Synthesis Inhibitors of Senescent Cells

C. Inhibition of DNA Synthesis by Poly(A)⁺ RNA

IV. Discussion

References

I. Introduction

Carcinogenesis has become widely accepted as a multistep process² (see References 1 and 2 for recent reviews). A number of events have to occur in order for cells to become cancerous. In many cases, if not all, cellular immortalization is one of these steps and is an obligatory process.³ Normal human diploid cells go through various numbers of population doublings (PDs), depending on the age of the donor and the origin of the tissue from which the cells are derived.^4–6 In most cases, cells from adult donors go through fewer PDs than cells from young or embryonic donors.⁶ The number of PDs that a culture can go through when derived from adult tissue is typically 20 to 30.⁵ Cancers generally are of clonal origin, and for a single cell to produce a tumor 1 g in size requires approximately 30 cell divisions. Primary tumors are not the major cause of problems in carcinogenesis because of the possibility of surgical removal of the primary tumor and, hence, the threat to the individual by that tumor. Indeed, metastasis is the crucial step that causes carcinogenesis to be a life-threatening phenomenon. Metastases are also generally of clonal origin and require a cell that has already gone through a number of doublings in the primary tumor to undergo further doublings as a metastatic growth in order to be significant. Cell growth, tumor regression, and cell death are all normal parts of the processes of carcinogenesis. Therefore, the number of PDs that cells have to go through in order to become life-threatening may be more than 100 to 200. This range is clearly greater than normal cells are able to go through, as evidenced by experiences with human fibroblasts in tissue culture.⁷ Other cells in the body may normally be able to go through more doublings in vivo. However, at this time, the doubling limit for most epithelial cells in situ is unknown. Therefore, it is reasonable to assume that as part of the multistep process of carcinogenesis, cellular immortalization is required for tumor progression and metastasis. The spontaneous immortalization of human cells in culture has never been observed. However, this can be contrasted with the situation that we see in rodent cells, particularly mouse and rat cells, in which spontaneous immortalization is the rule rather than the exception.^8–10 When considering whether immortalization may be necessary for tumor formation and metastasis, it is interesting to compare the rates of tumor formation in rodents with those in humans. A mouse weighs on the order of 10 g while humans weigh on the order of 100 kg, and the mouse’s life-span is approximately 1/30 that of a human, yet mice very often have tumors during their 2½- to 3-year life-span. Therefore, on a per cell unit time basis, the rate of tumor formation in mice is 10⁵ to 10⁶ times the rate of tumor formation in humans. It seems likely that this incredibly higher rate of tumor formation seen in mice compared to humans is due to the much higher incidence of spontaneous immortalization of mouse cells compared with human cells. Therefore, the study of cellular immortalization and of mechanisms that limit the proliferative potential of normal human cells in culture is of paramount importance in understanding the mechanisms of carcinogenesis in humans.

II. In Vitro Cellular Aging is Dominant in Somatic Cell Hybrids

A. Limited In Vitro Life-Span of Normal Cells

Swim and Parker were the first to show that human fibroblasts derived from biopsies had a limited proliferative potential in culture.¹¹ Hayflick and Moorehead in 1961 showed that these cells were karyotypically normal and that normal cells derived from a large number of different individuals all had a finite proliferative potential.⁴ They also showed that a major characteristic of cells that were able to divide indefinitely, i.e., transformed immortal cells, was an abnormal karyotype. In 1965, Hayflick proposed that the limited in vitro proliferative potential of normal human fibroblasts in culture was a manifestation of aging at the cellular level.⁵ More recently, it has been proposed by O’Brien et al.³ that limited proliferative potential of normal cells in vitro and also in vivo is a powerful tumor suppressor mechanism. The observation of limited proliferative potential of normal cells in culture has been repeated in hundreds of labs and thousands of cultures over the last 30 years.⁷ The proliferative potential of the cells depends on the age of the donor,⁶ species of the donor,¹² and the site of biopsy.⁶ Typically, human embryonic cells will undergo 50 to 80 PDs before growth cessation, although it has been reported that some cells are capable of going through approximately 100 PDs before proliferation stops.¹³ Cells from other species go through fewer PDs than those from humans, the exception being the Galapagos turtle.¹⁴ The number of PDs that the cells are able to undergo is correlated with the maximum life-span of the species.

Cells spontaneously immortalize at various rates, depending on the species of origin of the cells. Human cells and chick cells have never been observed to immortalize spontaneously, while rodent cells routinely immortalize in culture and species such as bovine immortalize spontaneously only rarely.¹⁵ The mechanisms that lead to limited in vitro proliferative potential of normal cells in culture is not understood. A number of investigations have been carried out over the past 30 years to measure various biochemical, metabolic, and structural parameters of these cells as they age in culture, and with very few exceptions, which will be discussed later, no changes have been observed that could account for the irreversible division cessation. Likewise, the process by which cells escape the finite proliferative potential and become able to divide without limit (immortalization) is not understood. In order to try to understand the mechanisms operating in these processes, we and others have undertaken a series of experiments discussed below.

B. Hybrids Between Normal and Immortal Cells

The early work of Littlefield suggested that the limited proliferative potential (the senescence phenotype) might be dominant in somatic cell hybrids between senescent cells and young proliferating cells.¹⁶ However, the evidence was not conclusive and the prevailing belief at that time was that cellular immortalization was due to dominant changes in the cellular genome. Many different ideas have been presented to try to explain the limited proliferation of normal cells in culture. These can be broken into two main categories. One category proposes that cells stop dividing because they accumulate damage of various sorts, e.g., somatic cell mutations or errors in protein synthesis, so that the error burden becomes so large that the cells are no longer able to divide. The other category proposes some sort of genetic program that limits the in vitro life-span of cells in culture. We thought that we might be able to differentiate between these two broad categories of hypothesis by fusing normal cells with immortal cells and determining whether the hybrids resulting from that fusion had a limited in vitro life-span or were immortal. If normal cells stopped dividing because they had accumulated a large amount of damage, then one could argue that cells that are immortal have escaped from limited proliferative potential because either they don’t accumulate damage at the same rate or they have evolved a mechanism to better cope with the damage. Therefore, one might expect in hybrids that the phenotype of cellular immortality would be dominant.

In the first set of fusions, we fused an immortal SV40-transformed cell line with a normal cell line that was at the end of its in vitro life-span.¹⁷ We observed that the hybrid colonies proliferated for various numbers of PDs and then stopped dividing. About 70% of the colonies were able to go through fewer than 8 PDs, while the other 30% were able to go through a range of PDs varying from 30 to 60, but they all stopped dividing. We also showed that all of the clones expressed the S V40 large T-antigen which is thought to be the immortalizing agent for normal human diploid fibroblasts infected with SV40 virus. In order to investigate the generality of this phenomenon, we fused a number of different cell lines with normal human diploid fibroblasts and observed the same results in all cases.¹⁸ The hybrids had finite proliferative potential. In all the fusion experiments, we found that immortal variants arose in the culture at a frequency of approximately 1 per 10⁵ to 10⁶ cells. This is a much greater frequency of immortalization than that observed in normal diploid fibroblasts. The tentative explanation for this is that in hybrids, chromosomal segregation takes place and the hybrids lose a chromosome which encodes a gene that causes the finite proliferative potential. Conclusions from these experiments are that the limited life-span of normal cells in culture is dominant over the phenotype of cellular immortality and that cells become immortal because they lose some of the program that is necessary to impose a limited proliferative potential on normal cells in culture.

C. Fusion of Immortal Cells with Other Immortal Cell Lines

If cellular immortality results from recessive changes in the cellular genome, then we might expect that different defects could occur to render a cell immortal. If that is the case, then fusion of cell lines having one defect with cell lines having another defect could result in complementation, giving a hybrid that has finite proliferative potential. On the other hand, fusion of cell lines having the same defect would not result in complementation and would give rise to hybrids that could divide indefinitely. Therefore, we would predict that hybrids resulting from fusions among different immortal cell lines would give two different kinds of results. In one case, some hybrids would have a finite life-span and the other hybrids would have an indefinite lifespan. In a series of experiments, Pereira-Smith and Smith fused different cell lines with each other and observed the proliferative phenotype (either finite or indefinite),¹⁹ and assigned more than 30 different cell lines to four different complementation groups. In order to begin the process of complementation group assignment...

Table of contents

• Cover Page
• Title Page
• Copyright
• Preface
• The Editors
• Contributors
• Table Of Contents
• Chapter 1 In Vitro Cellular Aging and Immortalization
• Chapter 2 Detection of Growth Factor Effects and Expression in Normal and Neoplastic Human Bronchial Epithelial Cells
• Chapter 3 Human Cell Metabolism and DNA Adduction of Polycyclic Aromatic Hydrocarbons
• Chapter 4 Human Esophageal Epithelial Cells: Immortalization and In Vitro Transformation
• Chapter 5 Transformation of Human Endometrial Stromal Cells In Vitro
• Chapter 6 Factors Influencing Growth and Differentiation of Normal and Transformed Human Mammary Epithelial Cells in Culture
• Chapter 7 Transformation of Colon Epithelial Cells
• Chapter 8 Multistep Carcinogenesis and Human Epithelial Cells
• Chapter 9 Morphologic and Molecular Characterizations of Plastic Tumor Cell Phenotypes
• Chapter 10 Oncogene and Tumor Suppressor Gene Involvement in Human Lung Carcinogenesis
• Chapter 11 Events of Tumor Progression Associated with Carcinogen Treatment of Epithelial and Fibroblast Compared with Mutagenic Events
• Chapter 12 Progression From Pigment Cell Patterns to Melanomas in Platyfish-Swordtail Hybrids — Multiple Genetic Changes and a Theme for Tumorigenesis
• Index