Table Of ContentVIROLOGY
MONOGRAPHS
DIE VIRUSFORSCHUNG
IN EINZELDARSTELLUNGEN
CONTINUINGjFORTFVHRUNG VON
HANDBOOK OF VIRUS RESEARCH
HANDBUCH DER VIRUSFORSCHUNG
FOUNDED BY j BEGRVNDET VON
R. DOERR
EDITED BY jHERAUSGEGEBEN VON
S. GARD • C. HALLAUER· K. F. MEYER
8
1971
SPRINGER-VERLAG
WIEN NEW YORK
SPONTANEOUS AND VIRUS
INDUCED TRANSFORMATION
IN CELL CULTURE
BY
].PONTEN
1971
SPRINGER-VERLAG
WIEN NEW YORK
All rights reserved
No part of this book may be translated or reproduced in any form
without written permission from Springer.V erlag
© 1971 by Springer.VerlagfWien
Softcover reprint of the hardcover 1st edition 1971
Library of Congress Catalog Card Number 75·137784
Printer: Elbemiihl, A·1230 Wien
ISBN·13:978·3· 7091·8260·4 e·ISBN·13:978·3· 7091·8258·1
DOl: 10.1007/978·3·7091·8258·1
Spontaneous and Virus Induced Transformation
in Cell Culture
By
J. Ponten
The Wallenberg Laboratory, University of Uppsala,
Uppsala, Sweden
With 35 Figures
Table of Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
A. Definitions of Transformation in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1. Irregular Growth Transformation ............ . . . . . . . . . . . . . . . . . . . 5
2. Unrestrained Growth Transformation ........................... 8
a) Non-specific Medium Depletion............................... 10
b) Cell Cycle Inhibition is Caused by Cellular Release of Factors
Inhibiting Cell Division ..................................... 11
c) Cell Cycle Inhibition is Triggered by Close Proximity (or Physical
Contact) between Cells without the Transmission of any Extra-
cellular Material ........................................... 12
3. Infinite Growth Transformation ................................ 15
B. Relationship between Transformation in vitro and Neoplasia in vivo.
Use of Implantation Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 16
1. Genetic Incompatibility ....................................... 17
2. Tumor-cell Antigens .......................................... 17
3. Size of the Implanted Dose of Cells ............................. 17
4. Site of Inoculation and Nature of the Tumors Formed ............ 17
5. Selective Pressure in vitro ..................................... 18
6. Induction of Neoplasia in the Host ............................. 18
C. The Nature of Transformations Involving the Control of Cell Growth 18
II. Spontaneous Transformation of Non-haemopoietic Cells. . . . . . . . . . . . . . . .. 20
A. Frequency of Spontaneous Infinite Growth Transformation in Different
Species ................•••..................................... 21
B. Biological Significance of Inter-species Differences. . . . . . . . . . . . . . . . . .. 25
C. Chromosome Analysis of Established Cell Lines ..................... 25
D. The Significance of Genetic Heterogeneity in Established Cell Lines .. 26
E. Cytology of Spontaneously Established Cell Lines ................... 27
F. Irregular Growth Transformation in Established Cell Lines ........... 28
Vlrol. Monogr. 8
2 J. Ponten: Spontaneous and Virus Induced Transformation in Cell Culture
G. Unrestrained Growth Transformation in Established Cell Lines ....... 28
H. Nature of Spontaneous Infinite Growth Transformation. Induction or
Selection ....................................................... 28
1. Antigenic Changes in Established Cell Lines from Normal Tissues .... 29
III. Spontaneous Lymphoblastoid Transformation of Human Lymphoid Tissue
or Peripheral Blood ................................................ 30
A. General Description of Lymphoblastoid Transformation. . . . . . . . . . . . .. 31
1. Lymphatic Tissue and Bone Marrow.. .. .. . . .. . .. . ... ... . . . . .... 31
2. Peripheral Blood ............................................. 32
3. Relationship between Lymphoblasts and Fibroblasts ............. , 35
B. Production of Immunoglobulins after Lymphoblastoid Transformation 35
C. Presence of Virus-like Particles in Lymphoid Lines .................. 37
D. Search for a Specific Antigen in Burkitt Lymphoma Lines ........... 39
E. Chromosomes and Lymphoblastoid Transformation ................. , 41
F. Mechanism for Lymphoblastoid Transformation - Selection or Induction 42
G. Relation between Lymphoblastoid Transformation and Neoplasia.... 42
IV. Virus Induced Transformation in Cell Culture. . . . . . . . . . . . . . . . . . . . . . . . .. 45
A. Transformation by DNA Viruses ...................... , ..... , ..... 46
The Papova- and Adeno Virus Groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 46
1. Polyoma Virus and Simian Virus 40 ............................ 46
a) Physico-chemical Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 46
b) Variants ............................................•..... 50
c) Virus-cell Interactions of PV and SV 40 ....................... 51
d) Lytic Effects of Polyoma and SV 40 .......................... 51
2. Transformation by Polyoma Virus .............................. 59
a) Individuality of Transformed Lines .......................... 61
b) Induction of Irregular and Unrestrained Growth Transformation. 61
c) Rate of Transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 64
d) Possible Causes for Cellular Resistance to Transformation ....... 65
e) Chromosomes and Polyoma Transformation.. ....... . ... .. . . .. 67
f) Tumorigenicity of PV Transformed Cells .. . . . . . . . . . . . . . . . . . . .. 68
g) Antigens in PV Transformed Cells. . . . . . . . . . . . . . . . . . . . . . . . . . .. 69
h) Attempt to Determine the Size of the PV Genome Necessary for
Different PV Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 72
i) Persistence of Polyoma Genome in Transformed Cells. . . . . . . . . .. 72
3. Transformation by Simian Virus 40 ............................. 75
a) Transformation of Stable Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 75
b) Transformation of Unstable Cells or Established Cell Lines. . . . .. 83
c) SV 40 Induced Antigens .................................... 85
d) The State of the SV 40 Genome in Transformed Cells ........... 87
4. Synopsis of Transformation by PV and SV 40 .................... 91
5. Transformation by Papilloma Virus ............................. 93
6. Transformation by Adenovirus ................................. 94
a) General Features of Adenovirus. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 94
b) Adenovirus Transformation in vitro .......................... 97
7. Transformation by Combinations of SV 40 and Adenovirus ......... 101
B. Transformation by RNA Viruses ............... " ........... , ..... 110
The Avian Leukemia-Sarcoma Complex ............................... 110
Table of Contents 3
1. Virological Aspects - Classification of Viruses ................... 110
a) Morphology of the Virions .................................. 110
b) Chemical Composition ...................................... 111
c) Viral Assembly ............................................ 113
d) Viral Envelope Constituents ................................. 113
e) Viral Core Constituents ..................................... 117
f) Origin of RSV Stocks ...................................... 119
g) The Species Range of RSV .................................. 119
h) Replication of Leukemia-Sarcoma Viruses in Avian Cells ....... 119
i) Subgroups of Viruses of the Avian Leukemia-Sarcoma Complex .. 122
j) Genetically Determined Cell Susceptibility .................... 122
k) Viral Interactions, Helper Effects ............................ 124
1) Infection of Other Bird Cells than Chicken Cells ............... 127
m) Infection of Mammalian Cells by RSV ........................ 128
2. Transformation fm, vitro by Members of the Avian Leukemia-Sarcoma
Complex .................................................... 129
a) Predominantly Sarcomagenic Virus Strains . . . . . . . . . . . . . . . . . . .. 129
b) Predominantly Leukemogenic Strains ........................ 135
c) Tumorigenicity of Transformed Cells ......................... 137
d) Relation between Virus Synthesis and Transformation .......... 139
e) Reversion and Loss of RSV Genome ......................... 140
f) Transmission of the RSV Genome . . . . . . . . . . . . . . . . . . . . . . . . . . .. 141
g) Metabolism of RSV Infected Cells . . . . . . . . . . . . . . . . . . . . . . . . . . .. 142
h) Avian Tumor Viruses and Differentiation ..................... 145
The Murine Leukemia-Sarcoma Complex .............................. 147
1. Pathology and Classification ................................... 147
2. Physico-chemical Properties of Virions .......................... 152
3. Immunological Properties of Mouse Leukemia Viruses ............. 155
4. Genetic Susceptibility to Murine Leukemia Viruses ............... 156
5. Murine Leukemia Viruses in Cell Culture ........................ 157
6. Murine Sarcoma Viruses in Cell C~ture ......................... 159
7. Interactions between Murine Leukemia and Sarcoma Viruses ....... 161
Synopsis of Transformation by RNA Viruses .......................... 165
V. General Survey and Discussion ...................................... 167
A. Characteristics of Transformed Cells ............................... 167
B. Mechanisms for Viral Transformation ............................. 176
1. Irregular and Unrestrained Growth Transformation ................ 177
2. Infinite Growth Transformation ................................ 184
3. Model of Virus Transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 184
Acknowledgements ..................................................... 186
References ............................................................. 186
1·
4: J. Ponten: Spontaneous and Virus Induced Transformation in Cell Culture
I. Introduction
A. Definitions of Transformation in vitro
When normal tissues or organs are explanted to conditions favoring the growth
of cells as individual units ("cell culture"), the original cell population undergoes
a large variety of modifications. Only a minority of the cells will thrive and
multiply and within a rather short period of time, the complex composition of
the original explant is replaced by a much simplified one of only a few recogniz
ably different cell types. With most organs fibroblast-like cells survive longest
and outgrow other types. This is then a stable state of affairs for many gener
ations. This treatise will not discuss whether this simplification and stabilization
represents selection of certain pre-existing cell types or a modification of cells
into only a few recognizably different categories; for an excellent review see
HARRIS. (1964).
Table 1. Terminology Employed to Describe Transformations in vitro
Type of transformation Essential features
Irregular growth Lack of contact inhibition of cell membrane movement
("ruffled membranes") between juxtaposed cells
Unrestrained growth Deficient inhibition of the cell cycle (mitosis) in a
crowded culture
Infinite growth Capacity of cells to undergo an infinite number of di
visions (formation of established cell lines)
Cells may depart from this typical behavior in numerous ways involving for
instance cellular morphology, immunology, chromosomes or metabolism. Such
changes have, sometimes rather vaguely, been called "transformations". This is
unprecise and the term "transformation" will here be used exclusively to indicate
disturbances in cell growth related to neoplasia. Reversible phenotypical alter
ations, for instance these dependent on a modified medium composition, will
not be considered.
Table 1 lists the three different types of disturbances in the regulation of
cell growth in vitro which havelbeen recognized:and defined as transformations
in this review. Although they may exist independently they are most often
combined.
The Tissue Culture Association has issued a Proposed Usage of Animal Tissue
Culture Terms (FEDOROFF, 1967). The recommended terminology has been followed
with one important exception. The T.C.A., proposed that 'the term "cell trans
formation" should be reserved to mean changes induced in the cells by the
introduction of new genetic material and that the new genetic material should be
specified'. They proposed that persistent changes in 'morphology, chromosome
constitution, virus susceptibility, nutritional requirements, proliferative capacity,
malignant character, etc.' should be termed culture alteration.
This definition of transformation is obviously inspired by~ bacterial gene
tics. Somatic cell genetics is, however, not yet sufficiently developed to meet
the requirements called for by an analogy with bacterial transformation. The
Introduction 5
definition of cell transformation by the T.C.A., read literally, would include the
effects of any lytic or non-lytic virus infection, since new genetic material would
be introduced into the cells. An artificial division would be created between for
instance SV 40 infected cells where there is definite evidence for the permanent
presence of newly introduced DNA which is transcribed and translated into
"T-antigen", and spontaneously altered mouse cells where there is no such evi
dence. Both cells have acquired the permanent changes listed in Table 1 and are
also tumorigenic after animal implantation. This review accepts that transforma
tion has a different meaning in bacterial genetics and in the study of animal cells,
which is in accord with the most common usage of the word.
1. Irregular Growth Transformation
The Lack of Oontact Inhibition of Oell Mernlwane Movement Makes Oells Oapable
ot Moving over each Other to Form Randomly Arranged Multilayers
The basis for this concept is an important observation by ABERCROMBIE and
HEAYSMAN (1953 and 1954) on the interaction of normal fibroblasts when they
come in close contact in vitro. Using time-lapse cinematography and direct obser
vation, these authors found that isolated chick heart fibroblasts showed irregular
movements of their cell membrane. When cells approached each other, membrane
movement was paralyzed along the line of contact and adhesions were formed. In
this way the cells ceased to be randomly oriented and became arranged as regular
parallel units. The phenomenon was called contact inhibition of locomotion. A
detailed and very informative intenerence microscope study of living fibroblasts
showed the undulating movement (ruffled membrane) to be confined to the lateral
cell borders (ABERCROMBIE and AMBRosE, 1958). Ruffling did not seem to be
solely responsible for net directional cell movement but was intimately con
cerned with pinocytosis. Contact inhibition of ruffled membranes explained why
cells move centrifugally from an explant and do not arrange themselves as a
pile of irregularly overlapping units, as would be expected if there were no restric
tions in cell movements (ABERCROMBIE and HEAYSMAN, 1954; CURTIS, 1961). Even
if contact inhibition seems to be the most important factor controlling cell
movement in vitro other phenomena such as contact guidance (HARBISON, 1914;
P. WEISS, 1934, 1945, 1958) and possibly also negative chemotaxis play a role.
However, ABERCROMBIE and GITLlN (1965) recorded the movement of small
groups of chick embryo heart fibroblasts and concluded that contact inhibition
rather than chemotactic gradients is the decisive factor in the determination
of cell locomotion.
In striking contrast to primary fibroblasts, sarcoma cells were found to have
lost their contact inhibition (ABERCROMBIE, HEAYSMAN, and KABTHAUSER, 1957).
When they approached each other or normal fibroblasts the ameboid movement
of the malignant cell's membrane continued and they moved freely on top of
the other cells. This produced a disorderly three-dimensional arrangement which
was easily distinguished from the regular two-dimensional pattern of normal
fibroblasts.
Contact inhibition and its absence have only been extensively studied in
populations of fibroblast-like cells (ELSDALE, 1968). Recent evidence (VAUGHAN
and TBINKAUS, 1966) suggests, however, that normal epithelium in culture is
6 J. Punten: Spontaneous and Virus Induced Transformation in Cell Culture
also subject to contact inhibition. Whether carcinoma cells in vitro display the
same lack of contact inhibition as sarcoma cells is unknown.
PONTEN, WESTERMARK, and HUGOSSON (1969) studied astrocyte-like cells
SHEIN, 1965) which form the predominant outgrowth from human brain. Cine
matographic recordings and light microscopic observations showed essentially the
same features as in fibroblast-like cells. Astrocyte-like cells moved by way of
ruffled membranes and were strongly inhibited after mutual contacts had been
established. Glioma cells differed by their deficient contact inhibition of cell
membrane movements.
Fig. 1. Normal bovine fibroblasts in a culture subjected to daily medium changes. The cells are ar·
ranKed in at least two clearly identified layers. Within each the nuclei lie in parallel array. The kind
of ordered nuclear overlapping illustrated should not be considered an indication of irregular growth
transformation (lack of contact inhibition). May-Griinwald-Giemsa. Appr. magn. x 800
Macrophages (amoebocytes) (CHANG, 1964; VmOLAINEN and DEFEND!, 1967)
may also survive long in culture but have not been studied with respect to cell-cell
interactions.
An ideal method for measuring the contact inhibition of cell locomotion and
membrane movement has not been found. The original and very reliable method
of direct observation supplemented by time-lapse cinematography (ABERCROMBIE
and HEAYSMAN, 1966) is not well suited for experiments on a large scale. A method
has been proposed whereby the growth pattern of large numbers of cells can be
evaluated in fixed preparations (ABERCROMBIE and HEAYSMAN, 1954). The number
of overlapping nuclei is compared with that expected for a random arrangement
of nuclei in the sample area. Later a modification of the original method was
introduced where the nuclei were idealized as ellipses rather than circles (CURTIS,
Introduction 7
1961}. The method avoids the time-consuming and subjective direct observations
of living cells under phase contrast but is not satisfactory because it fails to
differentiate between a completely disordered arrangement and cell sheets on
top of each other but with a normal array within each layer. The latter arrange
ment is observed among normal fibroblasts, particularly after prolonged culti
vation (cf. ABERCROMBIE, 1961), and scores significant nuclear overlapping, but
it probably does not reflect any disturbance of the normal contact inhibition
(ELSDALE and FOLEY, 1969). Figures 1 and 2 clarify the difference between the
two types of multilayering.
Fig. 2. Bovine fibroblasts after transformation by polyoma virus. The total cell density is about the
same as in Fig. 1. The essential difference is the complete randomness by which the transformed
cells are distributed in all three dimensions. This appearance is typical of irregular growth transforma-
tion (= lack of contact inhibition). May-Griinwald-Giemsa. Appr. magn. x 800
Cells may have suoh a strong adhesion to their plastic or glass support that
they remain anchored there in spite of undiminished membrane activity or
they may detach as soon as they reach the upper surface of a neighbouring cell.
Both these instances will show no nuclear overlapping in spite of a lost contact
inhibition of ruffled membrane movements. In this review, a requirement for
the acceptance of an irregular growth transformation will be a documentation
not only of an abnormally high cell density and growth in multilayers but also
a random arrangement of the cells in all three dimensions of the culture. Loss
of contact inhibition of membrane movement in a monolayer is only accepted on
cinematographic evidence_
The mechanism by which contacts induce membrane paralysis is unknown_ It
has been suggested that the surface charge is important (ABERCROMBIE and
