CURRENT CONCEPTS
OF
MALIGNANT GROWTH
Part A. From a Normal Cell to Cancer
by JĀNIS 0. ĒRENPREISS
CURRENT CONCEPTS OF MALIGNANT GROWTH
Part A. From a Normal Cell to Cancer
JANlS 0. ERENPREISS Latvian Institute of Experimental & Clinical Medicine, Riga, LATVIA
Edited by GUNTIS BRUMEUS and MARUTA DZERVE
1993
ZVAIGZNE PUBLISHERS RIGA, LATVIA
© Janis Erenpreiss, 1993
All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise without the prior written permission of the Author
ISBN—5—405—01282—3
Published by
Zvaigzne Publisher
Kr. Vademara iela 105,
Riga, LV 1013
LATVIA
Licence N 000304186.
Cover design by Arnolds KreslinshLayout design by Elizabete Gurska Desktop publishing by Maris Polkmanis
Printed in the Republic of Latvia
Preface 5
Abbreviations6
Chapter I. DEFINITIONS OF TERMS7
Chapter 2. CHEMICAL CANCEROGENESIS:
DISCOVERY OF THE BASIC PRINCIPLESOF CANCEROGENESIS13
General 13
Initiation .14
Promotion 16
The latency periodof tumour development21
The role of oncogenesin chemical cancerogenesis22
Chapter 3. THE ONCOGENE CONCEPT:
A HISTORICAL OVERVIEW 23
Discovery of oncogenes. The molecular mechanism of cancero genesis seems to be simple and clear 23
The oncogenes are numerous and diverse 25
Classification27
Distributionin living organisms 28
Locationon human chromosomes 32
Mechanisms of activation33
The sis oncogene: the last effortfor an unified concept 39
Chapter 4. THE ONCOGENE CONCEPT:
CURRENT INTERPRETATION43
Complementary oncogenes43
Oncogene expression in tumours46
The key oncogenes,myc and ras48
myc48
ras . . 54
General54
p2Iras59
Mechanisms of ras oncogene activation61
Protein-kinase and related oncogenes67
The abl oncogene ..67
src and related oncogenes69
A classification of oncogenes according to their functionin cancerogenesis 71
Some other oncogenes 73
Chapter 5. THE EMBRYOLOGICAL THEORY
OF CANCER: AN ATTEMPT TO COMPREHEND THE MULTITUDE OF DATA 79
From Julius Cohnheim to the oncogene 79
Gametogenesis: the role of oncogenes 85
Origin of germ cells 85
Female germ cell development88
Regulatory mechanisms of oocyte meiosis89
Male germ cell development97
Gene expression during spermatogenesis 98
Chapter 6. SYNOPSIS: CONCLUSIONS,SPECULATIONS, PREDICTIONS 103
References113
Preface
Theory provides a conceptual coherence for a certain field of knowledge. Factual data of this field serve as material for the construction of a theory.
Any attempt to develop a theory of cancerogenesis encounters a major impediment, which is not lack but plethora of data. That is why the first task is to select the appropriate data from this pool of oncological phenomenology.
During its century-long history, experimental oncology has arrived at certain statements which permit outlining of a general framework for the theory. Research in chemical cancerogenesis disclosed the most general rules of the conversion of a cell from normal into tumorous. The discovery of oncogenes, which followed and is continuing, revealed the molecular mechanisms of cancerogenesis.
However, extension of research, undertaken with great enthusiasm, yielded such a variety and diversity of cancerogens and oncogenes that the statements which were initially clear became blurred.
It is obvious that a guiding idea, a kind of working hypothesis that could serve as a criterion for selection of the basic data, is needed for outlining a general theory of cancerogenesis.
The guiding idea proposed in this book is as follows.
A normal biological process may provide a basic pathway for cancerogenesis. Thus, the key to understanding cancerogenesis is to be searched for in both identification and investigation of these normal pathways as tentative analogues of cancerogenesis. In my view, a molecular analogy between embryonal and cancer cells, i.e., the ability of any tumour cell to synthesize proteins of embryonal type, gives sound reasons for searching for normal analogues of cancerogenesis in the pathways of early ontogenesis.
These considerations gave an impetus for an attempt in this book to analyse contemporary data of experimental oncology from the viewpoint of the oldest concept of tumour growth, the embryological theory of cancer.
I should like to express my gratitude for the help and advice that several persons gave me during the making of this monograph. So, I should like to thank my colleages, Mrs. Taiga Gulite, Mrs. Ruta Zirne, and Mr. Maris Brazma, who helped me with the bibliographic reference system. Also, I note with appreciation the work done by Mr. Brazma in keyboarding the text and amendments.
J.O.Erenpreiss Riga, Latvia, March 1993.
AAP— 2-acetylaminophenanthrene
ASV— avian sarcoma virus
B(a)P, BP— benzo(a)pyrene
DMBA— 7,12-dimethylbenz(a)anthracene
EBV— Epstein-Barr virus
ENU— ethylnitrosourea
GF— growth factor
EGF— epidermal growth factor
FGF— fibroblast growth factor
PDGF— platelet-derived growth factor
TGF— transforming growth factor
IP3— inositol triphosphate
MCA— 3-methylcholanthrene
MNU— N-methyl-N-nitrosourea MSV— murine sarcoma virus
PAH— polycyclic aromatic hydrocarbon
PK-C— calcium activated, phospholipid-dependentprotein-kinase, protein-kinase C
RSV— Rous sarcoma virus
SV40— Simian virus 40
TPA— 12-0-tetradecanoylphorbol-13-acetate (alsocalled myristate acetate)
DAG— diacylglycerol
Chapter'
1
Definitions
of terms
A complete and widely recognized theory of cancerogenesis is lacking at present. Therefore, the interpretation of cancerogenesis terms is variable. It is extremely important to define the terms used here, at the beginning of this work. The definitions given below may or may not correspond to those given by other authors. It is most important that the reader clearly comprehends what the author wishes to express when using each term.
Cancerogenesis terminology also differs depending on the level being analysed: organism, cellular or molecular. The cellular level is given priority in this book when describing any events. However, an attempt is constantly made to describe any related molecular mechanisms. Most of the concise definitions given in this section will be expanded later with appropriate references. Only the literature which deals directly with the terminology of cancerogenesis is cited in this chapter.
Cancerogenesis is the standard term to describe generation of neoplasia in the broadest sense [28, 405]. In English, the term "carcinogenesis" is most often used [404, 461, 503]. This term in its strict sense signifies the development of an epithelial tumour only (a carcinoma). For this reason a group of experts [28] propose to replace the term "carcinogenesis" by "cancerogenesis".
Cancerogenesis is a specific biological process which is the same regardless of etiological agent, animal species, and reacting tissue.
For cells growing in vitro, the term "(malignant) transformation" is commonly used. This means that a cell line is capable of growth as a tumour when implanted in a suitable host [643].
Induction implies the generation of neoplasia that would not have occurred in the absence of an inducing agent [302].
It is necessary to outline the boundaries of cancerogenesis: it begins with the appearance of specific cellular changes and ends with the emergence of the first tumour cell. Cancerogenesis does not include normal development or pathology which precedes or follows malignization, such as progression, metastatic spread, differentiation, etc. Since cancerogenesis is treated as a cellular process, the metabolism of the cancerogen, for example, its activation, cannot be attributed to cancerogenesis, as it is sometimes done [289J.
No satisfactory definition for cancer (cell) has hitherto been suggested. Instead, multiple characteristics are proposed, which are for the most part optional (not indispensable). The most fundamental features of tumour cells are those designated by the term autonomy. This means that tumour cells do not behave as integral elements of the body, irrespective of whether they proliferate more or less rapidly than the normal cells, remain undifferentiated, or undergo terminal differentiation. "Autonomy’’ is sometimes equated with "independence", which is untenable both linguistically and oncologically (Greek "autos” - self, "nomos" - law, rule). The tumour cell autonomy is closely related to (yet not identical with) their autodynamic properties, viz. fixation of malignant properties, their persistence after removal of etiological agents, and transmission of these properties to subsequent cell generations. Fixation of malignant properties distinguishes tumorous from phenotypicalIy transformed cells. Phenotypically transformed cells may display the same features as tumour cells, but they return to the normal state as soon as the agent inducing these properties is removed.
The establishment of cell autonomy indicates that cancerogenesis is complete and a tumour has emerged.
Cancerogen is a chemical, physical or biological agent which induces cellular changes that are both specific and necessary for cancerogenesis.
Cancerogenesis has two stages, initiation and promotion. Accordingly, cancerogens fall into two groups, complete and incomplete cancerogens [461]. A complete cancerogen (synonym: solitary cancerogen) [28] is an agent that has the ability to induce the entire process of cancerogenesis [28, 155, 461]. An incomplete cancerogen is able to induce only one of the two stages. Hence, the incomplete cancerogens are subdivided, into initiators and promoters. Every division (group) includes chemical, physical, and biological agents. Practically all chemical and physical initiators act as complete cancerogens when applied at sufficient dosage, whereas biological initiators fail to do so. Under certain experimental conditions, some promoters behave as complete cancerogens of low potency.
Continuous application of a cancerogen causes tumours only after a certain minimum latent period. The formula that takes this principle into account is called the Weybull distribution [802].
The latency period of tumour development, or tumour latency [608], or induction period [302]is the time between the first application of a cancerogen and the emergence of a tumour [29]. The duration of
tumour latency is determined by the animal species, the cancerogen type, the experimental regime, and the cellular and molecular mechanisms of cancerogenesis. Shortest latency is determined exclusively by the cancerogenesis mechanism, and it is an inherited species-specific trait. Its length cannot be reduced by elevating the cancerogen dosage [928]. A reduced length of the shortest latency is possible when the cell omits some event(s) of cancerogenesis. This is possible if: (I) at the onset of experiment, the cell already possesses certain tumorous properties; (2) the cell genome becomes integrated with an activated oncogene; or (3) only phenotypic transformation but not actual cancerogenesis occurs.
Phenotypic transformation is a labile manifestation of certain tumorous cellular features that occur in response to extrinsic factors and disappear when the action of the agent is cancelled. It can be induced, e.g., by the transforming growth factor (TGF) that causes anchorage-independent cell growth [461].
Initiation is the first stage of cancerogenesis (both induced and spontaneous) [461]. Initiators are agents capable of inducing the first stage of cancerogenesis, the process of initiation [814]. The term "initiator" is sometimes misused as a synonym of "complete cancerogen" [62, 481]. Correspondingly, initiated cells are defined as cells capable of forming carcinomas when grafted into an animal [798]. Initiated cells do not exhibit tumorous phenotype.
Promotion is the second and final stage of cancerogenesis. A (tumour) promoter is an agent capable of inducing the second stage of cancerogenesis where a cell is converted from initiated to neoplastic [461]. Besides the above-mentioned (oncological) definition, the term "promoter" is used in biology to denote the site of transcription initiation on the DNA molecule.
Until 1976, promoters (croton oil, TPA) were commonly known as "cocancerogens" and the term "promotion" described the procedure of cocancerogen application [404]. Soon after, Berenblum substantially expanded the meaning of "cocancerogen" [77]. As a result, this term was in the latest terminology recommendations [28] deprived of semantic value and was hence abandoned from use in this book.
An oncogene is a gene, whose activity causes one of the stages of cancerogenesis. The terms, and corresponding symbols, used for designating oncogenes were proposed by J.M.Coffin et al. [173]: viral oncogene=v-onc; cellular oncogene=c-onc. The frequently used term, "protooncogene", denotes "the normal cellular counterpart of a gene identified as causing a tumour" [461]. However, ras seems to be the only gene for which a clear distinction can drawn between its activities as an oncogene and as a protooncogene. Therefore, it is not surprising that the two terms were freely interchanged when describing in the same paper both "the role of oncogenes in normal development" and "the expression of protooncogenes in tumour cells” [98]. I share R.A.Weinberg's view that "protooncogene" is merely an ill-made synonym of c-onc[1113], and hence I shall not use it in this book.
Oncogene activity implies three different meanings: (i) oncogene function, which like the function of any other structural gene is manifested through transcription and translation (expression). However, expression does not necessarily signify that the gene would function as oncogene in a given situation, since most, if not all, oncogenes fulfil physiological functions under certain conditions; (ii) the transforming activity of oncogenes, which can be detected in transfection assay. However, only some oncogenes possess this activity. The myconcogene, for example, cannot be assayed [8]. Consequently, a negative result of transformation test does not exclude the possibility that the tested gene is an oncogene, (iii) for the rasoncogene, the mutant allele is considered to be active.
Complementary oncogenes are pairs of oncogenes that jointly realize the entire process of cancerogenesis. Synonymous to the term "complementary oncogenes" is "cooperating oncogenes" [452, 548].
The protein encoded by an oncogene is designated "p" if it is exclusively the product of an oncogene, and "P", if it is a fused protein, i.e. the product of both an oncogene and an adjacent viral gene (gag, pol or env). Phosphorylated and glycosylated oncoproteins are abbreviated as "pp" and "gp", respectively.
Reference is made to "early event" and "late event" in speculations on the role played by oncogenes at any stage of cancerogenesis, especially when the time of activation of the ras oncogene is discussed. The "early" and "late" events, used in this context, are not attributed to any clearly-cut stages of cancerogenesis. This confusion in terminology is responsible for a good deal of controversy as to the role of the ras oncogene in the stages of cancerogenesis (for details, see Chapter 4.3.2.3.).
Much confusion has also been introduced by the use of the term "progression". Each author tends to attribute a personal meaning to this word, as, f.i., the following: the entire process of cancerogenesis [820]; a particular stage thereof, e.g., promotion [90]; a special case of cancerogenesis, e.g., neoplastic transformation in vitro [360]; or a third (after initiation and promotion) stage of cancerogenesis [295, 813]. The term "progression" was introduced by Foulds, who gave an unequivocal description of the process and its definition [302]. According to Foulds, progression means phenotypic and genotypic changes oftumour cells, transition from benign to malignant tumours and a rise in malignant properties [28, 302]. Since the emergence of tumour cells indicates the completion of cancerogenesis, it is clear that progression, which represents further multiple changes of the tumour that has already emerged, cannot be attributed to any period of cancerogenesis. Relations between cancerogenesis and progression are schematically depicted in Fig. I.
Fig. 1. The stages of cancerogenesis.
Besides the oncological terminology discussed above, some other terms that are often used in relation to cancerogenesis should be explained.
Gene regulation and also the regulation of oncogenes are governed by various mechanisms. Two types of interactions of genes within the same genome can be distinguished, cis- and trans-regulation [232].
Regulatory factors are called "trans-acting", if the genes that encode them do not reside on chromosomes containing the genes they control. The cis acting factors interact with "cis" recognition sequences, which are defined as the transcriptional apparatus elements residing beside the genes they regulate.
Fig. 2. Three types of intercellular regulation (stimulation): a = autocrine, b = paracrine, c = endocrine.
Molecular communication between cells occurs by autocrine, paracrine and endocrine regulation [461, 993,1055] (Fig. 2). Autocrineregulation is self-stimulation of a cell by production of both a stimulating
factor and its specific receptor. Paracrine regulation involves stimulation of a cell via the action of a substance produced by a neighbouring cell. Endocrine regulation denotes stimulation by a factor that is produced by a specific cell in a gland, and acts at a distance from this cell.
Cells belonging to the germ (cell) line give rise to spermatozoon and egg (ovum), and transmit genes from generation to generation and are therefore potentially immortal. All other cells are somatic.
A cell strain is a population of cells subcultured more than once in vitro, and it lacks the properties of indefinite serial passage. A cellline is a population of cells grown for an indefinite period of time in vitro by serial subcultures. This period of time presumes potential "immortality" of the cells when serially cultured in vitro [401],
chapter Chemical Cancerogenesis:
2
Discovery of the Basic
Principles of Cancerogenesis
2.1.GENERAL
Most of the present research in tumour development is directed to the study of oncogenes. However, the basic laws of cancerogenesis have been established by research of chemical cancerogenesis. These laws are as follows: stage-wise development; irreversibility; specific laws of tumour latency.
The two stages of cancerogenesis were discovered by Deelman [229, 230] who found that scarification of mouse skin pre-treated with coal tar stimulated the emergence of tumours that arose on the edges of the healing wound. A decade later, Twort and Twort [1079] induced tumours with oleic acid in mouse skin pre-treated with BP. Oleic acid was later substituted by croton oil [75].
The term "cocancerogen" was initially proposed to describe a secondary agent capable of inducing tumours only after application of a sub-threshold dosage of a cancerogen [404]. This term has now been replaced by "promoter". The notions of two-stage cancerogenesis were completely formulated by Berenblum and co-authors [76, 78, 80, 318, 461, and 928]. They argue that cancerogenesis in mouse skin consists of two stages, initiation and promotion. A complete cancerogen (for example, B(a)P) acts both as initiator and promoter when applied at sufficient dosage. However, only the initiating potencies are manifested when applied at sub-cancerogenic doses. Initiated tissues can develop tumours after application of a promoting agent. The promoting agents do not exhibit tumorigenic potency in noinitiated tissue.
The initiated state of a cell persists throughout the life of affected animals [250]. Further, after exposure of a pregnant P generation to an initiating dosage of a cancerogen, the cells of the Fl, and even F2, generations are promotable with TPA [679]. The laws of two-stage cancerogenesis, revealed by using B(a)P, extend to other cancerogens from the PAH-group, and other groups of chemicals [28]. Physical cancerogens also possess the initiation activity [308, 466, 683, 718J. Incomplete physical and chemical cancerogens are mutually interchangeable. For example, initiation can be induced by radiation, and subsequent promotion by TPA [685J. Two-stage cancerogenesis, which has been demonstrated primarily on mouse skin, can also be induced in other species, organs, and tissues [928]. The processes of initiation and promotion can also be reproduced in vitro [166, 414; 9641. Cells exposed to ENU transplacentally in vivo are promotable by TPA in vitro[520].