.. _ if. ...
m . mm... "EN. mw. .. ... .. .. ..
N M Du, an. m
.. c 8 Nu ...L . .. ..
x m .L. K _ . . .
W fi _
. 0” w to... . . .
m ....L N“
M B. ...C .. _
_ . .
~ .
._ . . -
w
. . 1
m .
_m . .
.w - .
p
p
p . . - .
., .
. .

Thesis for the Degree of ..M. S.

TY
ERLEYi.-'

o

STATE UNIVERSi

FREDERSCK ARTHUR wax

MICHIGAN

.

O

    

         

 

    

     

 

      

 

 

 

.. ._ . .4 .» .4... o.c..!o<‘¢‘g
.. . . ..
o . _ . . .4. .. . . .0. .....‘oofgoc 1|
. . . ..
. - . y
.,
o D O O.
O O
.
. . .—
._
.
o
_ .
.
o
n a 4 O
. . .
_ I
_
.
. 1.04.09“.
. . .. . .. 0 .,.4 .
. .v . -.....A.r:o.b...4l..4.. b
. c . . 4,, .4. . ......1.....»42_
. - . b . .9 .. . . o '1 O
. r . . . .... t. .3 .Jv. U c n
. ... _ . . ._ _ .4. v I . o .4.
. f . .. . . . . ...v ...... 4. ... . o .w:
a a . . . .
no 4
. . ....c... ..4.. VHS... 4...
. .
. . .. ..... ... 0. .-.. u
.
. . .. .... ..
.,. .. v
. .
d
. — .. a. ._
. _ .
. . . ...—-
u 4 0.
. .. ....
. 4 ......
_
. _ . . ..o:
. . Q n.
. . .. . _ .
. . .. . . .
. ....4
4 . .. .
. .. _ .
. . .
. ...... .
_ ... l.._.. JHMoPJ‘A
. ... .. _. . 0...“...
. . .. v... fl.“
. . o1. ... .0..4(44...
. _ . . . . . . ..4'...l..[ ’IO.
. ._ .o “.43. 4” .‘4.
. . . v . uh... .r0.Ao.h¢Yv 0V
. . . . ..-... 4 f O .
.0. .- . O4 . u . . . _|, ’4 I! 5.4.. — '4 W 0.4 4 ...“..O
... .. .<.. c I '0400.o...4\_’v. «.51
. . . . . . .. . or... .,. 4....0 .w-..1~ 0.4..
. 4 .. o .1.
v . 4. o. .
_ - . 9 ..
u. o O O. '
o A . . ......
. . . ....
C . . a -
. . . .. 4...
. .. . .o v ...;
. . . ,_ ..
. ..v 4.. .
.. _ _ , . .
. .4 . . ..
. . ... . .4.
A. a 4 ....
. . ...» v
- . I . ‘ .
. .4. ..I.. . ...... 04.:
. . . . ....'.4l.....\.u..r.l. I,4'o..ob ...v
. . . .. .. I...4v 2.4.1.- 0. .4..4 ..l
. . .. ... .0 00 . 04 n n. . . . .c'.. .' ..DVOJQIO o. _'
A . (LI-...... .~‘~Oo.ou'v
. . .4. .. .o., . coo .
0 4 . . . .4 .4 tn“! .V , 1.4 £( 54’.
. . . ... . . ..4 .14....19
. 4 . ... . .
IO .4... . ..Q .r....
. .. . . . . -
o . .4. . 4 . r: r
. . . 4 ..4. 4 a
. J 0.. .4 . ...”:onl
I . . .. .
4 . . . . .. .....I.....I.O..
. .. .
. I. A . a»
o ..
7 . . . . a ...0 o
. _ .. . —. .. -
_ . . . ..v... .
. o . . . . .lla.
. . . o .4. . 4:0...
. 4. . . . .. I. l ..04
a. .. . .......-
1 _ . .- .. . . .
. ... v r . . .
. .. . .4 . . 1..
I Q. l l
4 I ... I. O o C .. :04
. . .. . . o.
A . 4 .n ;
.o .4 . .... . .HJ...!: ..l
. . .. . . u I... A . .
. . . . o .0. .000... .
u I v. I a 14". up -"'0 c
-. 1
. . 4 . .“AIr . .
-. 1
r ‘4 .. 4 .o .4 O .rc.l
. . I. o 3. .. . o ... .0. o
.. . .. .. .l.
.. .4 v. . ... .. .... . .
. . 4 o .. . v I
. . . 4. . . .. I . 0.. . .04
0.. I.
.. a. 7 . ... . . .... ..l.. .
. . . .
0 c. 409.. ...... 0U..- 44. a. 00 ..
... . . .
. -r .. .v. . . ..
, . ..
. u I . . .l 4.....w. . .
_ .4 t . o . V- c. A. l
t v a
. . .
9‘. ..r. .
... o ...:
a » ...J. t
4 . I . .
. . O l—- O
. . o
o u I. O I 0.. 0|
I I I .
o . . 4 ..4 t‘ '4 ..- 0
.. . o
. 1! ... ... . .
. 4 . .0 0....
l U 0 .0-
. o
4 .4. . ..4
. u a o .. c o J. 4
. o .0 0 V a ‘o.
I On I U I .1 O r O
' 0 —
. 40....
_ .
. . . I. so
Cr 0 o I. I
- . ..
. . .44 . . A n
. . .... - . .n
. . .. o. n . .
. .r .
. . ... . 0 .
o o ..0 a .. ... I . .
. n . . O.
. . . r o. .t'rmol
. n . . ..o .
' . ... I. I
- O D A n
. I. . ...: .
.. . o 0.. c
I .. . o.
o
. . . .
..I O ’p t
. . .
. u o .444
. . a . 4 . cl .
4 I OI O 0 '4.ll O f
- 4 ' . u n 4
n o v : n. . t.
. .0). v u
n. c
.. . I . o
. . .. n. .. . .'I.:. 10
. -.. 7.1
. , Co v 70 a
. - n
A . a. . n. i .
o. . I.
4 nob—n .U'o ' O
. 4 .4
u I I. I. I o
. a 4
u . .0. I. O A l. .
. v I v at
4 . . .
l '0
4 . .
‘IO . ll .
0 0 I. J-
. .. .
. . .5.4 y. In. ul'.
l d
...
...; ad
.... .. .. ; . .........—... ......m...r Viv...)- . ...—$4.?J»...D. '34?
. .,__ . .. .. v. i... ... a I ...
......u. ............... .7... 104...... .... .....9.0 .W..:.....-.. :54
. r . . .
.

 

"ht-5‘5

 

   
    

V "F 3:3»an 3v 3
‘ “Ni-”'8“!
BUUK BINDERY W3
IJBRARY BINDERS
l.

2:335an. ”ICING I
_ “L‘: Ala-...-

  

 

  

ABSTRACT

BIOLOGICAL CHARACTERISTICS AND VIRAL SUSCEPTIBILITY
OF A CANINE KIDNEY ADENOCARCINOMA CELL LINE (CKT)

By

Frederick Arthur Wakerley

A cell line derived from a kidney adenocarcinoma of a 4-year-old
dog was serially cultivated in a closed system for 85 subcultivations
during a period of 2 years. The cells were epithelial-like in appear-
ance, had an abnormal karyotype, grew in soft agar, and exhibited
fluid transport activity, a function of secretory epithelium. Virus-
like particles similar to C-type oncornaviruses were observed budding
through and associated with the cell membrane. Growth of several

viruses was supported in the cells with resulting cytopathic changes.

BIOLOGICAL CHARACTERISTICS AND VIRAL SUSCEPTIBILITY

OF A CANINE KIDNEY ADENOCARCINOMA CELL LINE (CKT)

By

Frederick Arthur Wakerley

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1974

(3(1)? Gib

Dedicated to my wife
Cheryl -

and my father and mother

ii

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to all those who helped
with the completion of this study. I would especially like to thank
Dr. Gordon R. Carter and Mr. A. Wayne Roberts for their guidance
throughout this study and for their critical suggestions during the
preparation of this thesis. Grateful acknowledgement is also made to
Mr. Harold A. McAllister for his advice on photography, Mr. Stuart
Pankratz for his advice and assistance with the electron microscope,
and Dr. Alfred M. Legendre for help with and care of the animals used
in this study. I would also like to thank Mrs. Dorothy Boettger for
her efforts in securing necessary supplies.

I am indebted to Dr. Albert W. Dade of the Department of Pathology,
Michigan State University, for the histopathology, to Dr. Charles H.
Cunningham of the Department of Microbiology and Public Health, Michigan
State University, and Dr. Maurice Becker of the Michigan Department of
Public Health for supplying some of the viruses.

My wife Cheryl provided support and understanding that allowed me
to devote my energies to the completion of this degree. I sincerely
thank her and hope to compensate in the near future for all she has done.

This research utilized the Clinical Microbiology Laboratory
facilities and was supported in part by financial assistance from the
Department of Microbiology and Public Health.

iii

INTRODUCTION. . . . .

REVIEW OF THE LITERATURE.

TABLE OF CONTENTS

Development of Cell Culture. . .

Characteristics of Normal Cell Cultures.

Characteristics of Transformed Cell Cultures

Chromosome Analysis.

Species of Origin Determinations . . . . .

Oncornaviruses Associated with Cell Cultures

LITERATURE CITED. . . .

ARTICLE:

SUMMARY. . . . .

INTRODUCTION . .

MATERIALS AND METHODS.

RESULTS. . .

DISCUSSION . . .

LITERATURE CITED

iv

BIOLOGICAL CHARACTERISTICS AND VIRAL SUSCEPTIBILITY
A CANINE KIDNEY ADENOCARCINOMA CELL LINE (CKT).

Page

10

16

23
23
23
24
31
45

48

LIST OF TABLES

Table Page
1 Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . 28
2 Evaluation of mediums. . . . . . . . . . . . . . . . . . . 36
3 Comparative titrations of viruses in the CRT cell line

and in the donor host systems. . . . . . . . . . . . . . . 41

4 Results of cytotoxic-antibody dye-exclusion test for
species identification . . . . . . . . . . . . . . . . . . 42

LIST OF FIGURES
Figure Page

REVIEW OF THE LITERATURE

1 Fine structure of type A, B and C virus particles. . . . . 12
ARTICLE
1 Section of adenocarcinoma. Notice the tubule forma-

tion (arrOW) O O O O O O O O O O O O O O O O O O O O O O O 32

2 Section of adenocarcinoma. Notice the high mitotic
actiVity (arrOWS) O O O C O O O O O O O O O I O O O O O O O 32

3 Monolayer of CKT cells . . . . . . . . . . . . . . . . . . 33

4 Monolayer of CKT cells exhibiting "bleb" or dome
formation. 0 O O C O C C O O O O O O 0 O O O O O O O O O O 33

5 Growth curves of CKT cells at the 22nd (open circles)
and the 70th (closed circles) subcultures. . . . . . . . . 35

6 Growth efficiency of CKT cells. Curve indicates the
time required for development of confluent monolayers
at various seeding rates . . . . . . . . . . . . . . . . . 35

7 Chromosomal patterns of CKT cells at the 17th, 42nd
and 69th subcultures. Number in box represents the
number of metacentric or submetacentric chromosomes. . . . 37

8 Chromosomes of CKT cells. Notice the large number of
metacentric and submetacentric chromosomes . . . . . . . . 39

9 Normal karyotype of the male dog. From Hsu, T. C., and
K. Benirschke. 1967. Ig_An atlas of mammalian chromo-
somes. Springer-Verlag, NY. . . . . . . . . . . . . . . . 39

10 CKT cells infected with pseudorabies virus. (a and b)
- Notice the extensive syncytial formation (arrows);
(c) - Advanced infection. Polykaryocytes detached from
the substratum with balloon-like appearance. . . . . . . . 40

vi

Figure

11

12

13

Page

Growth of CKT cells in soft agar. Notice the vesicle
formation due to fluid transport activity (arrow). . . . . 43

Electron micrograph showing a Cetype virus particle
budding from the cytoplasmic membrane (arrow). . . . . . . 44

Electron micrograph showing C-type virus particles
outside the cytoplasmic membrane (arrows). . . . . . . . . 44

vii

INTRODUCTION

The in vitro cultivation of cells with stable characteristics is
a valuable tool in biological and medical research. Biochemistry,
microbiology, cancer research, and other fields of study have bene-
fited from the use of cell cultures.

Relatively few canine cell lines have been established and
described (2,35,48,68,74). A cell line derived from normal dog kidney
(MDCK) was established by Madin and Darby in 1958 (74). This line has
been widely used in animal virology (35). Rosanoff (68) established
a testicular tumor cell line, and Kasza (48) established cell lines
from a melanoma and a thyroid adenocarcinoma. The latter 2 lines had
chromosome alterations (62) similar to those described by Adams et a1.
(2) in cells derived from a canine venereal tumor. Miles et a1. (58)
reported similar findings in cells derived from canine lymphosarcomas.
Type-C virus particles have been associated with the canine venereal
tumor cell line (72).

The purpose of the present study was to describe the cultural
characteristics and viral susceptibility of a cell line derived from

a kidney adenocarcinoma of a 4-year-old dog.

REVIEW OF THE LITERATURE

Development of Cell Culture

 

Claude Bernard (9), in 1878, emphasized the importance of the
internal environment upon the regulatory activity of living tissue.
To attempt to study cells and how they affect or are affected by the
environment, he felt it was essential that cells be isolated in an
artificial system thereby avoiding the influence of the host. The
first steps made in this direction were made by von Recklinghausen
(64) in 1866 and Wilhelm Roux (70) in 1885, who were able to keep cells
and tissues alive in vitro. However, actual growth of cells was not
obtained. Beebe and Ewing (7), in 1906, using canine serum as a
medium, cultivated a canine lymphosarcoma, and Harrison (42), in 1907,
using nerve fibers, actually demonstrated cellular growth by embedding
tissue fragments in clotted lymph.

Considerable enthusiasm was generated by Harrison's work, but as
a result of frequent bacterial and fungal contamination, interest was
soon lost. Carrel, a surgeon and Nobel Laureate, in 1913 applied the
technique of asepsis introduced to surgery. Carrel's discovery in
1913 (18) that embryonic tissue extracts contain growth promoting
substances made it possible for the first time to propagate animal cells
indefinitely. He cultivated chicken fibroblasts using chicken plasma
as a nutrient, and was able to keep them alive for over 34 years. In

2

3
spite of the success of Carrel, tissue culture work was primarily con-
fined to bacteriologists and surgeons who were familiar with the rigid
demands of asepsis.

Early cell culture work employed nutritive media derived almost
exclusively from the organism and consisted of blood plasma, serum,
other body fluids and exudates, and extracts of tissues and organs.
The complexity and variability of these naturally occurring materials
made it difficult to use them in experiments designed to determine
nutritional requirements. It was not until the advent of antibiotics
in 1941 and the development of more refined mediums (5,41,60,82,85)
that tissue cultures became more widely used. In recent years chemi-
cally defined mediums of considerable complexity have been developed
(l4,24,63,80).

Earle (26), in 1943, described one of the first cell strains to
be established in continuous culture. The cells were derived from
normal areolar and adipose tissue of a mouse (L Strain). During the
1950's numerous cell lines from human and animal origins were established.
Gey et al. (37) established a stable cell line (HeLa) derived from a
human cervical carcinoma in 1952. Other cell lines derived from human
tumors were described by Fjelde (31), Berman and Stulberg (8), and
Eagle et al. (23). Hull et al. (45) established a cell line from a
Walker rat carcinoma in 1954.

Advances in tissue culture have greatly accelerated progress in
virology. In 1913, Steinhardt et al. (76) demonstrated that rabbit
cornea in plasma maintained the activity of vaccinia virus for more

than a month. Twenty-two years later, Rivers and Ward (66) carried out

4
vaccinia vaccinations using a strain of virus which had been passaged
for several years in vitro without intercurrent passages in animals.
Sabin and co-workers (71), in 1936, were able to grow poliomyelitis
virus in primary human embryonic neural tissue but not in other tissue.
In 1949 another major development occurred when Enders et al. (28)
grew the Lansing strain of poliomyelitis virus in human embryonic
tissue of non-neural origin. This finding, which marked the beginning
of modern virology, came at an opportune period in history. It was by
this time that the hazards of cell culture contamination had been

reduced due to the application of antibiotics.

Characteristics of Normal Cell Cultures

 

The development of improved methods for culturing animal cells in
vitro has greatly increased the usefulness of such cells. Microbiology,
pharmacology, pathology, immunology, genetics and biochemistry are a
few fields which have been advanced by cultivation of cells in vitro.

Tissues and organs are dissociated into cell suspensions by the
action of proteolytic enzymes such as trypsin (67). When inoculated
into a flask, the cells that grow out are called primary cells. The
monolayer thus formed will usually consist of fibroblast-like cells and
epithelial-like cells (19). Repeated subcultivation of a primary cell
culture usually causes selection for a certain cell type or types which
become predominant. These cells appear unaltered in morphologic and
growth properties. This diploid cell strain, as it is now called, must
be transferred at a relatively high cell density to initiate growth in
subcultures, and the cells have a limited life span, i.e., they cannot

be subcultured indefinitely. For example, WI-38, a human diploid cell

5
strain useful in isolating human viruses, has a finite lifespan of
approximately 50 population doublings (74).

The behavior of cells in organisms and cell cultures suggests that
normal cells are inherently able to regulate their growth. This
ability is manifested in vitro as postconfluence inhibition of cell
division (25). The various kinds of intercellular interactions which
could trigger postconfluence inhibition of cell division fall into 3
categories which are not mutually exclusive.

1. Metabolic cell-cell contact. This hypothesis states that
there is a direct contact of metabolically active cells. An inhibi-
tory substance is then transferred from one cell to another via membrane
junctions, without traversing the external medium (79).

2. Mechanical cell—cell contact. This hypothesis proposes that
inhibition may be caused by changes in cell shape, changes in area
exposed to the medium or mechanical restraints upon cell movement (56).

3. Local diffusion gradient. Inhibition, as stated in this
hypothesis, is caused by the establishment of a local diffusion gradient.
Local concentrations of an inhibitor or local depletion of a substance
necessary for cell division (or both) would trigger the mechanism for
postconfluence inhibition (65). The "wound healing" phenomenon as
described by Martz and Steinburg (56) provides evidence against a
uniformly distributed inhibitor. Wound healing is initiated when a
small patch of cells is scraped away from a stationary phase monolayer.
Almost all cells near the edge of the scrape divide within a few days,
but very few cells away from the scrape divide during this period.

Thus, a very high mitotic rate and a very low mitotic rate exist side

6
by side in the same culture, which cannot be explained with a uniformly
distributed inhibitor.
Occasionally during the multiplication of a diploid strain a few
cells may exhibit altered morphology, increased growth rate, and
increased growth efficiency. The clone derived from such cells has

unlimited life and constitutes a permanent cell line.

Characteristics of Transformed Cell Cultures

Cell lines repeatedly transferred undergo changes due to the emer—
gence and selection of variants. These cell lines occasionally are
neoplastic and are said to have undergone a "transformation" to the
neoplastic state (33). Cells may be transformed in viva or in vitra.
Histologic indications of in viva transformation are: dysplasia,
anaplasia, hyperplasia, increased nuclear—cytoplasmic ratio, and mitotic
aberrations (75). Malignant neoplasms show varying degrees of invasive-
ness. The ultimate invasive potential is the metastatic or secondary
growth (22).

There are several criteria for the determination of neoplastic
transformation in vitra. The 3 most important are production of a
malignant neoplasm in viva by inoculation of cells into a syngeneic
host, growth in soft agar, and immortality. Not all criteria for trans-
formation need be met; some transformed cell lines do not produce neo-
plasms when inoculated in viva (22). Normal cells in tissue culture
fail to divide when suspended in fluid or semisolid medium. Apparently
they require attachment and extension on a solid substratum in order to
divide (52). Virtually without exception all transformed cells that

grow in soft agar will produce neoplasms when inoculated into a suitable

7
host (22). Neoplastic transformed cells may also exhibit different
morphologic and growth characteristics when compared with normal cells
in vitra. Examples are loss of postconfluence inhibition of cell
division and increased saturation density (22). This relates only to
fibroblast-like cells grown in vitra; transformed epithelial-like
cells exhibit no morphologic alterations in many instances.

A number of vegetable proteins (lectins) such as wheat germ
agglutinin, concanavalin A (con A), and phytohemagglutinin bind in
vitra to carbohydrates on the surface of transformed cells but not
normal cells. These polyvalent lectins will agglutinate the transformed
cells in vitra. When mixed with normal cells no agglutination occurs
(47). Normal cells treated with trypsin, a protease, will agglutinate
with con A (46). Therefore, it is assumed that untransformed cells
contain the same or similar sites on the cell surface as transformed
cells but in a cryptic form. Transformed cells can be restored to
normal growth characteristics by covering the agglutinin sites with
monovalent con A obtained by treatment with trypsin (l6).

Transformed cells may also show an altered karyotype. A culture
that has acquired abnormal genetic characteristics but cannot produce
tumors in animals is referred to as having undergone genetic

transformation (33).

Chromosome Analysis

 

The relationship between chromosomal alteration and change from
normal somatic cells into cancer cells is an important area of cancer

research. While many investigators believe that chromosomal

8
alteration is only a secondary event in tumor induction, others believe
it plays an important role.

Boveri (11), in 1914, assumed that abnormal mitosis, i.e., irregu-
lar distribution of chromosomes frequently found in tumor cells, pro-
duced the potentiality for unrestricted cell proliferation. This
hypothesis was suported by Makino et a1. (54) and others.

In general, some deviations from normal somatic cells are found
in the chromosome complement of cells in primary and transplantable
tumors. In most tumor types, the site and type of alteration in karyo-
type differ from one tumor to another (87), while in others, e.g., the
canine venereal tumor (54), certain characteristic karyological changes
are exhibited. On the other hand, in the majority of mouse leukemias
produced with leukemogenic viruses, diploid karyotypes are retained
(87). Takayama (81) and Makino (53) have suggested that each tumor
population is characterized by the possession of a stemline karyotype
deviating from that of normal cells. In their opinion, the genetic
character of a tumor population is closely correlated with the chromo-
some constitution of its stemline cells. The karyotype of stemline
lineage, however, is not always stable, and the stem cells are easily
replaced by more vigorous cell types resulting in changes in stemline
karyotype.

The canine venereal sarcoma exhibits a similarity in stemline
karyotype. Various researchers have observed a modal number of 59
chromosomes, of which 16 or 17 are metacentric or submetacentric
(2,6,54,72,81). This represents a wide deviation from the normal dog

karyotype of 78 chromosomes with all the autosomes being acrocentric

9

(44). The consistency of similar chromosomal alterations in canine
venereal sarcomas has led some investigators to postulate that this
tumor type originated from a single dog (53,54,81). However, this
"Adam and Eve" hypothesis has been questioned since similar alterations
have been observed in cell cultures of canine lymphosarcomas (58), a
melanoma, and a thyroid adenocarcinoma (62).

Miles et al. (58) suggested that centric fusion, a process whereby
2 acrocentric or telocentric chromosomes fuse to form a single meta-
centric chromosome, was a possible mechanism to explain the increase
of metacentric and submetacentric chromosomes. That centric fusion does
occur was substantiated by Allerdice and co-workers (3) working with

mouse cell lines.

Species of Origin Determinations

Numerous methods have been developed for determining the species
of origin of cell cultures. Some of these methods are hemagglutina-
tion (12), mixed agglutination (21), cytotoxic-antibody dye-exclusion
(39), immunofluorescence (78), chromosome analysis (20), and isoenzyme
characterization (59). The confirmation of species of origin is
important as it is not uncommon for laboratory contamination of one
cell type with another to occur. Franks at al. (32). utilizing the
mixed agglutination test, found 29 cell lines contaminated with cells
of other species. The cytotoxic-antibody dye-exclusion test (39)
showed 3 cell types thought to have originated from porcine, chicken
and monkey tissues to actually be human cells. Immunofluorescence,
employed by Simpson and Stulberg (73), found 2 out of 26 cell lines to

contain cells of species other than originally designated.

10

Chromosomal analysis of mammalian cell lines has also been used as
an aid in the identification of the species of origin (20). Clausen
and Syverton observed that the chromosomes of 10 altered lines with the
supposed species of origin of cynomolgus monkey, laboratory rabbit,
porcine, and bovine resembled human chromosomes. They also observed
that 4 lines of supposed human origin and one of monkey origin possessed
chromosome complements resembling the L-strain of mouse origin. These
results were corroborated by immunologic experiments of Brand and
Syverton (l3) and was consistent with the virus susceptibility and
cell morphology.

It is also possible that a cell line of one species may contaminate
aqf overgrow a different cell line of the homologous species. For
example, Gartler (34) observed that 20 human cell lines, presumably of
Caucasian and Negro origins, contained a variant of glucose-6-phosphate
dehydrogenase (G6PD). He suggested that these were contaminated with
HeLa cells since this variant of G6PD occurred in only 30% of the Negro
race and never in Caucasians. Isoenzyme electrophoresis (59) confirmed
Gartler's experimental results and was consistent with his diagnosis.
These findings indicate the importance of proper intraspecies as well

as interspecies identification of cells.

Oncornaviruses Associated with Cell Cultures
Considerable attention in cancer research has been directed to the
finding of C-type, RNA containing virus particles in cell lines under
continuous culture (4,15,29,30,35,43,49,57,72,86). These virions are
enveloped, approximately 100 nm in diameter, and appear spherical to

pleomorphic. Assembly of the virion is at the cytoplasmic membrane

11
through which it buds. The core of C-type virions may be electron dense
or electron-lucent (Figure l). C-type particles contain 2 major anti-
gens, an envelope antigen and an internal or group specific (gs) antigen.
Two distinct gs antigens are found in the same protein chain; gs l,
which is species specific and does not cross react with viruses of other
species, and gs 3, which is common to all mammalian viruses (38).

Other oncornaviruses, classified by morphology, are A- and B-type
particles (Figure 1). Type A particles have an electron-lucent core
surrounded by an electron dense area. The nucleocapsid is centrally
located within a single membrane and the complete virion has the appear-
ance of a "doughnut." It is approximately 90 to 120 nm in diameter,
found in great numbers in the Golgi area, and may form inclusion bodies
visible in the light microscope (10). The significance of A-type
particles is as yet unknown. Type B particles differ from the C-type
particles of leukosis viruses by their dense eccentric core and 2
envelopes which are separated by an e1ectron-1ucent space (17). Type
B particles are variable in size, range from 90 to 150 nm in diameter,
and occasionally reach 200 nm. The envelope surrounding the core is
apparently derived from the endoplasmic reticulum, while the envelope
of the virion is formed from elements of the cytoplasmic membrane (17).
An example of B-type particles is the mouse mammary tumor virus which
is transmitted through the milk of infected mothers (51).

Ellermann and Bang (27), in 1908, discovered the first tumor virus
which causes leukemias in chickens. In 1911, Rous (69) described the
cell-free transmission of a tumor-producing agent. This discovery

demonstrated that solid tumors, in addition to leukemias, could be

12

 

Fine structure of type A, B and C

Figure 1.
virus particles.

13
caused by viruses. Manaker and Groupé (55), in 1956, observed that
Rous Sarcoma virus caused cells in culture to grow into localized foci.
This observation served as a starting point for the development of a
tissue culture assay for the virus (83).

Until 1951, leukemia viruses had not been demonstrated in animals
other than chickens. Cross (40), in that year, succeeded in passing
mouse leukemia from one mouse to another with cell-free extracts.

In 1973 Lieber et a2. (49), using the supernatant reverse tran-
scriptase assay, examined 31 cell lines of various species from the
American Type Culture Collection. Readily detectable amounts of
oncornaviruses were demonstrated in l pig, 2 Chinese hamster, and 3
rat cell lines. A fluorescent antibody technique was utilized by
Ferrer et a1. (30) to detect C—type particles in cell lines derived
from leukemic cows. The electron microscope was used by Armstrong
at al. (4) to detect C-type particles in pig cell lines, and by Sapp
and Adams (72) in a canine venereal tumor cell line.

Burger and Noronha (15) purified infectious C—type particles from
a cell line derived from a leukemic cat. When high concentrations of
such particles were inoculated into young dogs, a disease similar to
that seen in the cat was produced. Furthermore, the feline gs antigen
was detectable in the dog cells.

Lowryet aZ.(50) studied the activation of murine leukemia virus
(MLV) in cell lines from embryos of the high leukemic AKR mouse. Two
cell lines were negative for MLV as determined by extensive testing
for infectivity, viral antigens, morphologic particles, and reverse

transcriptase activity. However, on rare occasions, a spontaneous

14
activation estimated at a frequency of 10-8 or 10-9 would occur. An
exposure of these cells to 5—iododeoxyuridine (IDU) or 5-bromodeoxy—
uridine (BDU) induced as many as 0.5% of the cells to produce MLV, an
increase of about 106 over the spontaneous rate. Aaronson at al. (1)
exposed 13 non-virus producing clones of the mouse BALB/c embryo line
to BDU, which induced the production of C-type virus particles.

A further modification of the above technique involved the use of
dimethyl sulfoxide (DMSO) (77). Dimethyl sulfoxide was added to a
human tumor cell line previously activated by IDU and a tenfold increase
in observable virus particles occurred over treatment with IDU alone.

A recent hypothesis proposed by Todaro and Huebner (84) states
that vertebrates contain the genetic information for producing a C-type
virus in an unexpressed form in every somatic and germ cell. Further-
more, they suggest that this genetic information has been part of the
genetic makeup of cells since early in evolution and can persist for
hundreds of generations in cell culture without production of virus.
The endogenous "virogene" (genes for production of virus) and the
"oncogene" (part of the virogene, responsible for transformation to a
tumor cell) are maintained in an unexpressed form by unknown repressors
in normal cells. Derepression of the repressor substance would allow
expression of the genes. Vertical transmission by the germ cells is
the usual mode of transmission. The work previously described of Lowry
et a1. and Aaronson at al. (50,1) with DNA analogs have added substance
to the oncogene hypothesis since nonproducing clones have been induced

to form C-type particles.

15

An alternate hypothesis put forth by Temin (82) states that RNA
tumor virus replication is via a DNA intermediate which can be trans-
mitted in cells as DNA. The DNA intermediate, called a provirus, is a
template for the production of progeny viral RNA. This protovirus
hypothesis is consistent with the oncogene hypothesis in the contention
that the tumor cell has regions of DNA specifying synthesis of RNA that
leads to transformation, but this DNA is created by successive RNA to
DNA to RNA information transfers. The provirus is not present in germ
cells but is generated as the animal develops. Thus only a small frac-
tion of somatic cells would contain all the genes necessary for C-type

virus production.

LITERATURE CITED

10.

11.

LITERATURE CITED

Aaronson, S. A., Todaro, G. J. and E. M. Scolnick. 1971. Induc-
tion of murine C-type viruses from clonal lines of virus-free
BALB/3T3 cells. Science 174:157-159.

Adams, E. W., Carter, L. P. and W. J. Sapp. 1968. Growth and
maintenance of the canine venereal tumor in continuous culture.
Cancer Res. 28:753-757.

Allerdice, P. W., Miller, 0. J., Miller, D. A., and D. Warburton.
1973. Chromosome analysis of two related heteroploid mouse
cell lines by quinacrine fluorescence. J. Cell Sci. 12:
263-274.

Armstrong, J. A., Porterfield, J. S. and A. T. deMadrid. 1971.
C-type virus particles in pig kidney cell lines. J. Gen.
Virol. 193195-198.

Baker, L. E. 1936. Artificial media for the cultivation of
fibroblasts, epithelial cells and monocytes. Science §§g605.

Barksi, G., and Cornefert-Jensen, Fr. 1966. Cytogenetic study
of sticker venereal sarcoma in European dogs. J. Nat. Cancer
Inst._3l:787-797.

Beebe, S. P., and J. Ewing. 1906. A study of the biology of
tumor cells. Brit. M. J. 251559.

Berman, L., and C. S. Stulberg. 1956. Eight culture strains
(Detroit) of human.epithelial-like cells. Proc. Soc. Exp.
Biol. Med. 92:730-735.

Bernard, C. 1878. In_Lecons sur les phénoménes de la vie communs
aux animaux et aux végétzux. Paris.

Bernhard, W. 1960. The detection and study of tumor viruses with
the electron microscope. Cancer Research 295712—727.

Boveri, T. 1914. In_Zur Frage der Entstehung Maligner Tumoren.
Gustav Fischer, Jena, German. 64.

16

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

17

Brand, K. G., and J. T. Syverton. 1960. Immunology of cultivated
mammalian cells. 1. Species specificity determined by hemag-
glutination. J. Nat. Cancer Inst. 24:1007—1019.

Brand, K. G., and J. T. Syverton. 1962. Results of species
specific hemagglutination tests on "transformed", nontrans-
formed, and primary cell cultures. J. Nat. Cancer Inst. 28;
147—157.

Bryant, J. C., Schilling, E. L., and W. R. Earle. 1957. Massive
fluid-suspension cultures of certain mammalian tissue cells.
I. General characteristics of growth and trends of population.
J. Nat. Cancer Inst. 213331.

Burger, C. L., and F. Noronha. 1970. Feline leukemia virus:
purification from tissue culture fluids. J. Nat. Cancer Inst.
455499-503.

Burger, M. M., and K. D. Noonan. 1970. Restoration of normal
growth by covering of agglutinin sites on tumour cell surface.
Nature (London) 228:512-515.

Bykovsky, A. F., Miller, G. G., Yershov, F. I.,.Ilyin, K. V., and
V. M. Zhdanov. 1973. B-type oncornaviruses isolated from
continuous human cancer cell lines. Archiv. fUr die gesamte
Virusforschung 42;21-35.

Carrel, A. 1913. Artificial activation of the growth in vitra
of connective tissue. J. Exper. Med. §§g407.

Carter, G. R., and A. W. Roberts. 1973. Some methods employed in
the study of viruses. 'In_0utline of veterinary virology.
Lucas Brothers, USA. 8-19.

Clausen, J. J., and J. T. Syverton. 1962. Comparative chromosomal
study of 31 cultured mammalian cell lines. J. Nat. Cancer

Coombs, R. R. A., Daniel, M. R., Gurner, B. W., and A. Kelus.
1961. Recognition of the species of origin of cells in culture
by mixed agglutination. I. Use of antisera to red cells.
Immunology 4555-66.

Discussion Group 14. 1973. Criteria of neoplastic transformation.
Conference on carcinogenesis testing in the development of new
drugs. National Academy of Sciences, National Research Council.

Eagle, H. 1955. Propagation in a fluid medium of a human epi-
dermoid carcinoma strain KB. Proc. Exp. Biol. Med. 823363-364.

Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures.
Science 130:432.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

18

Eagle, H., and E. M. Levine. 1967. Growth regulatory effects of
cellular interaction. Nature 213:1102—1106.

Earle, W. R. 1943. Production of malignancy in vitra. IV. The
mouse fibroblast cultures and changes seen in the living cells.
J. Nat. Cancer Inst. 4:165.

Ellermann, V., and 0. Bang. 1908. Experimentelle Leukamie bei
Hflhmern. Centr. Bakteriol. Parasitenk. 46:595-609.

Enders, J. F., Weller, T. H., and F. C. Robbins. 1949. Cultiva-
tion of Lansing strain of poliomyelitis virus in cultures of
various human embryonic tissues. Science 109:85-87.

England, J. J., Watson, R. E., Jr., and K. A. Larson. 1973. Virus—
1ike particles in an equine sarcoid cell line. Am. J. Vet. Res.
3451601-1603.

Ferrer, J. F., Avila, L., and N. D. Stock. 1972. Serological
detection of type C.viruses found in bovine cultures. Cancer
Res. 3251864-1870.

Fjelde, A. 1955. Human tumor cells in tissue culture. Cancer
§g845-851.

Franks, D., Gurner, B. W., Coombs, R. R. A., and R. Stevenson.
1962. Results of tests for the species of origin of cell lines
by means of.the mixed agglutination reaction. Exp. Cell Res.
283608-612.

Freeman, A. E., and R. J. Hueber. 1973. Problems in interpreta-
tion of experimental evidence of cell transformation. J. Nat.
Cancer Inst. 59;303-306.

Gartler, S. M. 1968. Apparent HeLa cell contamination of human
heteroploid cell lines. Nature 217:750-751.

Gaush, C. R., Hard, W. L., and T. F. Smith. 1966. Characteriza—
tion of an established line of canine kidney cells (MDCK).
Proc. Soc. Exp. Biol. Med. 122:931-935.

Gazzolo, L., Simkovic, D., and M. C. Martin-Berthelon. 1971. The
presence of C-type.RNA virus particles in a rat embryo cell

line spontaneously transformed in tissue culture. J. Gen. Virol.
125303-311.

Gey, G. 0., Coffman, W. D., and M. T. Kubicek. 1952. Tissue
culture studies of the proliferative capacity of cervical
carcinoma and epithelium. Cancer Res. 12:264.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

19

Gilden, R. V., and S. Oroszylan. 1972. Group—specific antigens of
RNA tumor viruses as.markers for subinfectious expression of the
RNA virus genome. Proc. Nat. Acad. Sci. USA 62:1021-1025.

Greene, A. E., Coriell, L. L., and J. Charney. 1964. A rapid
cytotoxic antibody test to determine species of cell cultures.
J. Nat. Cancer Inst. 32:779-786.

Gross, L. 1952. Mouse leukemia. Ann. N.Y. Acad. Sci. 5451184—
1196.

Harris, M. 1952. The use of dialyzed media.for studies in cell
nutrition. J. Cell. Comp. Physiol. 495279.

Harrison, R. G. 1910. The outgrowth of the nerve fiber as a mode
of protoplasmic movement. J. Exp. 2001. 2:787.

Hooks, J., Gibbs, C. J., Chopia, H., Lewis, M., and D. C. Gajdusek.
1972. Spontaneous transformation of human brain cells grown
in vitra and description of associated virus particles. Science
116:1420-1422.

Hsu, T. C., and K. Benirschke. 1967. In An atlas of mammalian
chromosomes. Springer-Verlag, New York. Folio 20.

Hull, R. N., Cherry, W. R., and I. S. Johnson. 1956. The adapta-
tion and maintenance of mammalian cells ts continuous growth in
tissue culture. Anat. Rec. 124:490.

Inbar, M., and L. Sachs. .1969. Interactioncfifthe carbohydrate-
binding protein concanavalin.A with.normals and transformed
cells. Proc. Nat. Acad. Sci. 63:1418-1425.

Inbar, M., and L. Sachs. 1969. Structural difference in sites on
the surface membrane of normal and transformed cells. Nature
(London) 223:710-712.

Kasza, L. 1964. Establishment and characterization of canine
thyroid adenocarcinoma and canine melanoma cell lines. Am. J.

Lieber, M. M., Benveniste, R. E., Livingston, D. M., and G. J.
Todaro. 1973. Mammalian cells in culture frequently release
type C viruses. Science 182:56-59.

Lowry,D. R.,Rowz, W. P., Teich, N., and. J. W. Hartley. 1971.
Murine leukemia virus: High frequency activation in vitra by
5-iododeoxyuridine and 5—bromodeoxyuridine. Science 174:155-156.

Lyons, M. J., and D. H. Moore. 1965. Isolation of the mouse
mammary tumor virus: Chemical and morphological studies. J.
Nat. Cancer Inst. 35:549.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

20

Macpherson, I. 1969. Agar suspension culture for quantitation
of transformed cells. Habel and Selzman (eds.). Fundamental
techniques in virology. Academic Press. pp. 214-219.

Makino, S. 1963. Some epidemiologic aspects of venereal tumors
of dogs as revealed by chromosome and DNA studies. Ann. New
York Acad. Sci. 108:1106-1122.

Makino, S., Sofuni, T., and S. Takayama. 1962. Cytological
studies of tumors.. XXXIX. A further study of the chromosomes
in venereal tumors of the dog. The Nucleus 5fllS-122.

Manaker, R. A., and V. Groupé. 1956. Discrete foci of altered
chicken embryo cells associated with Rous sarcoma virus in
tissue culture. Virology 2:838-840.

Martz, E., and M. S. Steinberg. 1972. The role of cell-cell
contact in "contact" inhibition of cell division a review and
new evidence. J. Cell. Physiol. 123189—210.

McAllister, R. M., et al. 1972. C-type virus released from cul-
tured rhabdomyosarcoma cells. Nature New Biol. 235:3-6.

Miles, C. P., Moldavannu, G., Miller, D. G., and A, Moore. 1970.
Chromosome analysis of canine lymphosarcoma: Two cases involv-
ing probable centric fusion. Am. J. Vet. Res. 31:783-789.

Montes De Oca, F., Macy, M, L., and J. E. Shannon. 1969. Iso-
enzyme characterization of animal cell cultures. Proc. Soc.
Exp. Biol. Med. 132:462-469.

Morgan, J. F., Morton, H. J., and R. G. Parker. 1950. Nutrition
of animal cells in tissue culture. I. Initial studies on a
synthetic medium. Proc. Soc. Exp. Biol. Med. 13:1.

Oshimura, M., Sasaki, M., and S. Makino. 1973. Chromosomal banding
patterns in primary and transplanted venereal tumors of the dog.
J. Nat. Cancer Inst. 51;1197-1203.

Pakes, S. P., Griesemer, R. A., and L, Kasza. 1965. Chromosome
analysis of 2 canine tumor cell lines. Am. J. Vet. Res. 26:
837-843.

Puck, T. T., Cieciura, S. J., and A. Robinson. 1958. Genetics
of somatic mammalian cells. III. Long term cultivation of
euploid cells from human and animal subjects. J. Exp. Med.

108:945.

Recklinghausen, F. D., van. 1866. Ueber die Erzeugung von rathen
Blutkfirperchen. Arch. Mikroskop. Anat. 2;137.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

21

Rein, A., and H. Rubin, 1968. Effects of local cell concentra-
tions upon the growth of chick embryo cells in tissue culture.
Exp. Cell Res. 42:666-678.

Rivers, T. M., and S. M. Ward. 1935. Jennerian prophylaxis by
means of intradermal injections of culture vaccine virus. J.
Exp. Med. 62:549.

Roberts, A. W., and G. R. Carter. 1972. Cell culture procedures.
22 Simplified and economical cell culture techniques for labora-
tories beginning clinical veterinary virology. Michigan State
Agricultural Experiment Station No. 5677. pp. 3-25.

Rosanoff, E. I. 1959. Continuous cultivation of malignant cells
from a canine testicular tumor. Proc. Soc. Exp. Biol. Med.
100:384-387.

Rous, P. 1911. Transmission of a malignant new growth by means
of a cell-free filtrate. J.A.M.A. 56:198.

Roux, W. 1885. Beitrage zur Entwicklungsmechanik des Embryo.
Ztschr. Biol. 22:411.

Sabin, A. B., and P. K. Olitsky. 1936. Cultivation of poliomyeli-
tis virus in vitra in human embryonic nervous tissue. Proc.
Soc. Exp. Biol. Med. 24:357-359.

Sapp, W. J., and E. W. Adams. 1970. C-type viral particles in
canine venereal tumor cell cultures. Am. J. Vet. Res. 21:
1321-1323.

Simpson, W. F., and C. S. Stulberg. 1963. Species identification
of animal cell strains by immunofluorescence. Nature 199:

Shannon, J. E., and M, L. Macy (ed.). 1972. 22_The American type
culture collection registry of animal cell lines. 2nd ed.
American Type Culture Collection, Rockville, MD.

Smith, J. C. 1971. Introduction to neoplasms. 22_Pathology.
Peery, T. M., and F. N. Miller (eds.). pp. 329-348.

Steinhardt, E., Israeli, C., and R. A. Lambert. 1913. Studies
on the cultivation of the virus of vaccinia. J. Infect. Dis.
13:294.

Stewart, S. E., Kasnic, G., Jr., Draycott, C., and T. Ben. 1972.
Activation of viruses in human tumors by 5-iododeoxyuridine and
dimethyl sulfoxide. Science 175:198-199.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

22

Stulberg, C. 8., Simpson, W. F., and L..Berman.. 1961. Species-
related antigens of mammalian cell strains as determined by
immunofluorescence. Proc. Soc. Exp. Biol. Med. 108:434-439.

Subak—Sharpe, H., Bark, R. R., and J. D. Pitts. 1969. Metabolic
co-operation between biochemically marked mammalian cells in
tissue culture. J. Cell Sci. 4:353-367.

Swim, H. E., and R. F. Parker. 1958. Stable tissue culture media
prepared in dry form. J. Lab. Clin. Med. 22:309.

Takayama, S. 1958. Existence of a stemrcell lineage in an infec-
tious venereal tumor of the dog. Jap. J. Genetics 23:56-64.

Temin, H. M. 1972. The RNA tumor viruses - background and fore-
ground. Proc. Nat. Acad. Sci. USA 62:1016-1020.

Temin, H. M., and H. Rubin. .1958. Characteristics of an assay
for Rous sarcoma virus-and Rous sarcoma cells in tissue culture.
Virology 6}669-688.

Todaro, G. J., and R. J. Huebner. 1972. The.vira1 oncogene
hypothesis: New evidence. Proc. Nat. Acad. Sci. USA 69:
1009-1015.

White, P. R. 1946. Cultivation of anima1.tissues in vitra in
nutrients of precisely known constitution. Growth 293231.

Woods, W. A., Papas, T. S., Hirumi, H., and.M. A. Chirigos. 1973.
Antigenic and biochemical characterization of the C-type
particle of the stable porcine kidney cell line PK-15. J.
Virology 2251184-1186.

Yosida, T. H. 1968. Relationship between chromosomal alteration
and development of tumors in viva and in vitra. 22_Cancer cells
in culture. H. Katsuta (ed.). University Park Press, USA.
pp. 171-194.

ARTICLE

BIOLOGICAL CHARACTERISTICS AND VIRAL SUSCEPTIBILITY

OF A CANINE KIDNEY ADENOCARCINOMA CELL LINE (CKT)

By

F. A. Wakerley, A. W. Roberts, and G. R. Carter

(Manuscript to be submitted to the American
Journal of Veterinary Research)

SUMMARY

A cell line derived from an adenocarcinoma of a kidney from a
4-year-old dog was serially cultivated in a closed system for 85 sub-
cultivations during a period of 2 years. The cells had an abnormal
karyotype and exhibited fluid transport activity, a function of secretory
epithelium. Virus-like particles similar to C-type oncornaviruses were
observed budding through and associated with the cell membrane. Growth
of several viruses was supported in the cells with resulting cytopathic

changes.

INTRODUCTION

The in vitra cultivation of cells with stable characteristics is
a valuable tool in biological and medical research. Biochemistry,
microbiology, cancer research, and other fields of study have benefited
from the use of cell cultures.

Relatively few canine cell lines have been established and
described (1,5,7,20,22). A cell line derived from normal dog kidney
(MDCK) was established by Madin and Darby in 1958 (22). This line has
been widely used in animal virology (3,5). Rosanoff (20) established
a testicular tumor cell line, and Kasza (7) established cell lines from
a melanoma and a thyroid adenocarcinoma. The latter 2 lines had chromo-
some alterations (17) sindlar to those described by Adams et a2. (1) in
cells derived from a canine venereal tumor. Miles at al. (15) reported
similar findings in cells derived from.canine lymphosarcomas. Type-C

23

24
virus particles have been associated with the canine venereal tumor cell
line (21).
The present study describes the characterization and viral suscepti-
bility of a cell line derived from a kidney adenocarcinoma of a 4-year-

old dog.
MATERIALS AND METHODS

Origin and establishment af the CKT cell line. The CKT cell line
was established by one of us (AWR) from a tumorous kidney of a male
4-year-old Miniature Poodle. The cells were dispersed by overnight
trypsinization (19) (ca. 16 hours) and cultured in a closed system
using Eagle's minimum essential medium with Earle's base supplemented
with 0.5% lactalbumin hydrolysate, 1 mM sodium pyruvate and nonessential
amino acids (modified EMEM) with 10% bovine fetal serum (BFS). Potassium
penicillin G, streptomycin sulfate, and polymyxin B sulfate were added
at the rate of 150 units/ml, 150 ug/ml and 7S units/m1, respectively.

The cells were subcultured at 1—week intervals at a split ratio of
l to 3 bottles for the first 5 subcultures. The subculture interval
was extended at the sixth subculture to 5 weeks due to a reduced growth
rate, and the split ratio was decreased to 1 to 1. Medium was replaced
at 1-week intervals. By the 9th subculture, the growth rate had
increased and the split ratio was extended to 1 to 3. Thereafter, the
cells were routinely subcultured at 7- to 10-day intervals without a
change of medium. Cells were preserved in liquid nitrogen at the 15th
and 50th subculture. The methods used for subculturing and preserving

cells were as described elsewhere (19).

25

Glass and plastic ware. Water dilution bottles (Kimble, Toledo,
Ohio) fitted with plastic screw caps were used for stock cultures of
cells, and l-milliliter glass ampules (Weston Glass Company, Millville,
New Jersey) were used for the freezing and storage of cells. Disposable
30-m1 plastic flasks (Falcon Plastics, Los Angeles, California) were
used for growth rate, growth efficiency, and medium evaluation. Dis-
posable 16 x 15 mm plastic petri dishes (Falcon) were used for soft agar

suspension cultures.

Growth rate. The 22nd subculture and the 70th subculture were used
in the growth rate study. Five-milliliter aliquots of cell suspensions

4 cells/ml

containing 5.5 x 104 cells/m1 (22nd subculture) and 5.0 x 10
(70th subculture) were seeded into disposable plastic flasks and incu—
bated at 36 C. Quadruplicate counts of 2 flasks at each subculture
level were made in a hemacytometer at 12-hour intervals for the first
4 days and thereafter at 24-hour intervals. The growth medium was

replaced at 4-day intervals, and the experiment was terminated when the

number of cells decreased or detached from the flask.

Growth efficiency. The growth efficiency determination was per-

formed on the 70th subculture: 5.0 m1 cell suspensions of 2.2 x 105,

1.1 x 105, 5.5 x 104, 2.2 x 104, 1.1 x 103 cells/ml were seeded into
disposable plastic flasks. Two flasks for each dilution were employed

and medium was replaced at 4-day intervals. The time required for con-

fluent monolayers to form was recorded.

Evaluation of’growth and maintenance mediums. The mediums used

for the growth and maintenance evaluation were Eagle's minimum essential

26
medium (EMEM), modified EMEM, Earles' basal medium (BME), BME diploid,
medium 199 (Mr199), McCoy 5A, and Hanks' balanced salt solution supple-
mented with 0.5% lactalbumin hydrolysate and 0.05% TC yeastolate (LHY
Hanks'). The mediums were obtained commercially (International Scien-
tific, Cary, Illinois) as a prepared powder except for LHY Hanks' which
was prepared in the laboratory. Growth mediums contained 10% BFS and
maintenance mediums contained 2% BFS. Antibiotics as described earlier
were employed. Mediums were added to disposable plastic flasks in 4.5
m1 amounts. Subsequently, 0.5 m1 of a cell suspension of 6.0 x 105
cells/m1 was added. Five flasks were employed for each medium, and the
mediums were replaced at 4-day intervals. When a monolayer was formed,
the growth mediums were replaced with maintenance mediums in 3 of the
flasks; the growth mediums in the remaining 2 flasks were left unchanged.
Observations were made as to the period of time cells retained their
healthy appearance. Degeneration was the criterion used in the

determination.

Chromosome analysis. Chromosome analysis was performed on the 17th,
42nd, and 65th subculture of CKT cells. The method as described by
Merchant et al. (14) was used to obtain metaphase preparations which

were stained with a modified Giemsa stain (18).

Viral susceptibility. The 65th subculture and several subsequent
subcultures of CKT cells were used for viral susceptibility testing.
Cell cultures employed for the propagation of the stock viruses were pig
kidney [PEN (1)], African green monkey kidney (Vero), canine kidney

(MDCK), human cervical carcinoma (HeLa), equine kidney (EK), and bovine

27
kidney (BK). Avian viruses were cultivated in chicken embryos (CE).
The viruses tested and the host systems used for their cultivation are
tabulated (Table 1). Cell cultures were initiated as described else—
where (19); the medium used was the same as previously described for
CKT cells.

Stock viruses were diluted 1:10. Two tubes of CKT cells were used
for each virus and the inoculum was 0.2 ml/tube. The cells were incu-
bated at 36 C and observed each day for cytopathologic effects (CPE).
When CPE was evident (2+ or greater) or at 7 days postinoculation,
cells and medium were frozen, thawed and reinoculated into CKT cells.
At least 3 passages were made with each virus. Cytopathologic effects
and fluorescence microscopy, where applicable, were the criteria used
for determining infectivity.

Viruses that did not produce CPE in CKT cells were inoculated back
into the original host systems to determine if they had grown without
producing observable effects. Viruses that propagated in CKT cells
were comparatively titrated in the original host systems used for

propagating the viruses and in CKT cells.

Culturing of cells in soft agar. The method of Macpherson (10)
was used with a slight modification, the only change being the use of
20% BFS with modified EMEM. Disposable plastic petri dishes were
seeded with 5.5 x 104 cells/petri dish. Controls employed were the
MDCK cell line and a rat embryonic skin cell line (RESI) (8). The
cultures were incubated at 36 C in an atmosphere of 5% CO2 and air at

80% humidity. They were examined at 12 days and 18 days after seeding.

28

Table l. Viruses

 

Cell culture or Viral titer
animal source (TCIDSO/ml except
Virus of stock virus where indicated)

 

ANIMAL VIRUSES

Pseudorabies PEN (1) 5.0
Vesicular stomatitis (Indiana) PEN (1) 8.0
Hog cholera PEN (1) 4.5
Equine rhinopneumonitis (A-163) EK 3.5
Eastern equine encephalomyelitis Vero 7.5
Western equine encephalomyelitis Vero 5.5
Equine cytomegalovirus (82-A) EK 3.0
Equine adenovirus BK 2.5
Bovine adenovirus type 3 (WBRI) BK 4.0
Infectious bovine rhinotracheitis BK 5.5
Bovine virus diarrhea BK 5.0
Canine herpesvirus MDCK 1.5
Canine adenovirus type 2 (Manhattan) MDCK 7.0
Canine SV5 (958) MDCK 7.0
AVIAN VIRUSES
Newcastle disease virus (Roakin) CE (AC)* 6.5 (LDSO/ml)
Infectious laryngotracheitis CE (CAM)** 4.5 (IE50/m1)
Fowl pox CE (CAM) 6.0 (ID50/m1)
HUMAN VIRUSES
Adenovirus type 1 (AD-71) Vero 5.5
Poliovirus type 2 (Sabin) Vero 8.5
Herpes simplex (MacIntyre) HeLa 5.5
Coxsackievirus A16 Vero 4.5

Coxsackievirus B2 Vero 6.5

Table 1 (continued)

29

 

Virus

Cell culture or Viral titer

animal source
of stock virus

(TCIDSO/ml except
where indicated)

 

Coxsackievirus B5
Coxsackievirus B6
Echovirus 9 (Hill)
Reovirus 1

Reovirus 2

Vero
Vero
Vero
HeLa
HeLa

6.5
5.5
4.5
4.5
3.5

 

Data are expressed as reciprocal of log of virus dilution.

*
Allantoic cavity.

**
Chorioallantoic membrane.

30

Tumarigenic capabilities. The 67th subculture of CKT cells was
used to evaluate tumorigenicity in viva. The cells were washed 3 times
and resuspended in Hanks' balanced salt solution (388). Three 5-week-
old mixed breed dogs (littermates) were separately inoculated with
25 x 106 cells subcutaneously (SQ), intraperitoneally (IP), and intra-
muscularly (IM). One additional littermate was inoculated IP with
50 x 106 cells and IM with 25 x 106 cells. Six weanling Syrian hamsters
were inoculated with 1 x 106 cells; 3 were inoculated IP and 3 were
inoculated via the cheek pouch.

The dogs were housed in concrete runs and fed a standard diet.
The hamsters were maintained in standard cages (3 per cage). Immuno-
suppressants were not administered. The dogs and hamsters were sacri-

ficed 150 days after inoculation.

Species of origin determination. Hyperimmune anti-canine serum
was prepared by inoculating a New Zealand White rabbit with normal
primary canine kidney cells. The inoculum, consisting of 2.0 m1 of
an emulsion of cell suspension (106 cells/ml) and equal parts of
Freund's complete adjuvant (Difco, Detroit, Michigan) was administered
subcutaneously at 4 sites on the rabbit's back. Twelve days later,

1 m1 of the cell suspension without adjuvant was injected intravenously.
The rabbit was bled 13 days later and the serum was collected and
inactivated for 20 minutes at 60 C.

Confirmation of species of origin of the CKT cells was determined
by employing the immunofluorescence technique of Stulberg et al. (25)
with slight modifications, and the cytotoxic-antibody dye-exclusion

test as described by Greene et al. (6). The immunofluorescence

31
technique was modified by using the antiserum described above in an
indirect test rather than the direct test as described. Antirabbit
globulin conjugate was obtained commercially (Sylvana, Millburn, New
Jersey) and used at a 1:4 dilution. Vero cells were employed as a
control in the immunofluorescence test. Vero and MDCK cells were used

as controls in the cytotoxicity test.

Sterility tests. The 24th and 73rd subcultures were checked for
contaminating microorganisms. Sabouraud's dextrose agar, blood agar,
and mycoplasma broth and plates were inoculated with antibiotic free
CKT cell suspensions. Filtered CKT fluid from the 84th subculture was
inoculated into MDCK cells and underwent 2 blind passages. The 8lst
subculture of CKT cells was used for electron microscopic examination.
The procedure used was similar to that described elsewhere (23). Grids

were examined in an HU—ll Hitachi electron microscope at 75 KV.

RESULTS

The CKT cells were derived from a tumorous kidney diagnosed histo-
pathologically as an adenocarcinoma (Figures 1 and 2). They were
fibroblastic in appearance during the first 5 subcultures. At the 6th
subculture the growth rate of the cells diminished considerably. A
few areas of epithelial cells were observed at the 7th subculture, and
by the 9th subculture the cells were entirely epithelial-like in appear-
ance (Figure 3). "Blebs" or "domes", raised areas of cells one cell
layer thick, were observed as early as the 18th subculture and remained

evident through 85 subcultures during a period of 2 years (Figure 4).

32

 

Figure 1. Section of adenocarcinoma. Notice
the tubule formation (arrow). Hematoxylin and eosin
stain; X 1280.

 

Figure 2. Section of adenocarcinoma. Notice
the high mitotic activity (arrows). Hematoxylin and
eosin stain; X 1280.

33

 

Monolayer of CKT cells. Giemsa's

Figure 3.

stain; X 640.

.. .- 1:,
avg/mew.
. 21)..» /,. :
(1 o

.1;

 

Monolayer of CKT cells exhibiting

Figure 4.
"bleb" or dome formation.

Unstained; X 123.

34
These blebs attained a size up to 4 mm in diameter and were visible
macroscopically.
The growth rate of the CKT cell line is shown graphically (Figure
5). The mean doubling time for exponential growth was determined

using the equation:

Nt = ZktNo

where No is the population size at a certain time, Nt is its size at
a subsequent time, t, and k is the exponential growth rate constant,
defined as the number of doublings per unit time. The mean doubling
time is the reciprocal of the exponential growth rate, which was 36
hours for the 70th subculture and 45 hours for the 22nd subculture.
The cells detached from the surface at a stationary density of 3.0 x
105 cells/cm2 during the 70th subculture, and reached a stationary
density of 3.8 x 105 cells/cm2 during the 22nd subculture before the
cell number decreased.

A monolayer was formed within 2 days in the growth efficiency
test (Figure 6) when the cells were seeded at a concentration of 2.2
x 105 cells/ml, and 8 days when the cell concentration was 1.1 x 104
cells/ml. When seeded at 1.1 x 103 cells/m1, 22 days were required for
a monolayer to form.

The results of CKT growth in selected mediums are presented
(Table 2). Modified EMEM, EMEM, McCoy 5A, and LHY Hanks' supported
the growth of CKT cells; other mediums did not.

The chromosome complement of CKT cells at 3 subculture levels is

shown graphically (Figure 7). Fifty metaphase figures were counted for

 

 

 

35

 

. x
medium changes

 

 

 

_l
E
\
cr
LU
(D
2
D.
z
9
O
O
.1
_3 W
5 2 4 6 8 1O 12
TlME(days)

Figure 5. Growth curves of CKT cells at the
22nd (open circles) and the 70th (closed circles)
subcultures.

 

 

 

 

x .X X X
y medium changes

2001 1
3’
.\150
”s
.5

100-
co
m
w
a)
2
:3
Z 50‘
..l
.1
w
0

1 I T I v I I v 1 v 1 1‘7 1
O 4 12 16 2O

8
TIME (days)

Figure 6. Growthefficiencyof CKT cells.
Curve indicates the time required for development
of confluent monolayers at various seeding rates.

36

Table 2. Evaluation of mediums

 

Monolayer formation,
medium supplemented with

Degeneration of mono-
layer (days)

 

 

10% BFS (days) 2% BFS* 10% BFS**
Modified EMEM 3 11 ll
EMEM 3 12 7
BME none -- ——
BME Diploid none —— _-
M-199 none —— --
LHY Hanks' 3 12 8
McCoy 5A 3 8 8

 

*
After formation of a monolayer serum content was changed to 2%

and replenished every 4 days.

*
Medium not replenished after a monolayer formed.

 

37

102

86

82

78

   
  
  
   

N
o 3‘

S}

a:
N

Y.‘

NUMBER OF CH ROMOSOM ES/CELL

0'1
O

42

30

01234

NUMBER OF CELLS

7“"

Figure 7. Chromosomal patterns of CKT cells at the 17th, 42nd
and 69th subcultures. Number in box represents the number of meta-
centric or submetacentric chromosomes.

38
each subculture. Most of the cells of the 22nd subculture contained
chromosomes clustered in a range of 42 to 54 chromosomes per cell,
whereas chromosomes of the 42nd subculture were spread over a wider
range. The 69th subculture contained chromosomes which were again
clustered, but in a higher range of 63 to 74 chromosomes per cell. A
high frequency of metacentric and submetacentric chromosomes were
observed at all passage levels (Figure 8). The karyotype of CKT cells
differed markedly from that of normal dog cells, which have a diploid
number of 78 chromosomes per cell with only the X and Y being meta-
centric (Figure 9).

Of 27 viruses tested, the CKT cell line was susceptible to 8
(Table 3). The viral CPE produced in CKT cells was similar to that
produced in cell cultures normally used to propagate the viruses,
except for pseudorabies virus, which produced extensive syncytia
resulting in extremely large polykaryocytes (Figure 10). These poly-
karyocytes contained more than a hundred nuclei in some instances.
Viruses that grew in CKT cells did so, in most instances, to an extent
comparable with their propagation in the original host systems (Table 3).

The CKT cells were not adequate for the initial propagation of
the other 20 viruses tested; equine rhinopneumonitis was adaptable.
Eastern equine encephalomyelitis (EEE) produced CPE initially but not
in subsequent passages or when dilute inocula were used. No evidence
of infection was observed when these viruses were inoculated back into
the original host system.

Cell aggregates in soft agar were not observed immediately after

seeding. Examination of cells at 12 days revealed that approximately

39

'-

‘ OI r
u“ '* .-

..
.‘ 1° 7° 1““ .l
35‘2") . NW ' I."

o
. Ca. ‘

 

 

Figure 8. Chromosomes of CKT cells. Notice
the large number of metacentric and submetacentric
chromosomes. Giemsa's stain; X 3,200.

M II II u u u n
u u n u n In as
n u M u on an n
u u u u an u an

; Cl .- .0 On I. I. A.

q 5“ no 4. '-
1 x I

Figure 9. Normal karyotype of the male dog.
From Hsu, T. C., and K. Benirschke. 1967. _I_n_An
atlas of mlian chromosomes. Springer-Verlag, NY.

 

 

 

Figure 10. CKT cells infected with pseudorabies virus. (a and
b) - Notice the extensive syncytial formation (arrows); (c) - Advanced
infection. Polykaryocytes detached from the substratum with balloon-
like appearance. (a) - Giemsa's stain; X 640, (b) - Unstained; X 112,
(c) - Unstained; X 112.

41

Table 3. Comparative titrations of viruses in the CKT cell line and
in the donor host systems

 

Titration in CKT

 

cell line Titration in original
(TCIDSO/ml) host system (TCID50/m1)

Pseudorabies 6.0 5.0

Vesicular stomatitis 8.0 8.0

Canine herpesvirus 3.5 1.5

Canine adenovirus type 2 4.0 7.0

Canine SVS 5.5 7.0

Herpes simplex 7.0 5.5

Reovirus l 5.5 4.5

Reovirus 2 4.0 3.5

 

Data are expressed as reciprocal of log of virus dilution.

42

60% of the CKT cells had divided with 40% having too great a number to
count (Figure 11). Cell division in the positive control (RESI) was
80% with most aggregates (70%) containing too many cells to count. In
contrast, there were only a few aggregates of cells (5%) observed in
the negative control (MDCK) and these only contained 2 and 4 cells.

The CKT cells were confirmed to be of canine origin. Peripheral
fluorescence was observed with CKT cells but not with Vero cells. The

results of cytotoxic-antibody dye-exclusion test are presented (Table 4).

Table 4. Results of cytotoxic-antibody dye-exclusion test for species

 

 

identification
Preinoculation serum Anti-normal dog serum
(viability) (viability)
MDCK 91% 0%
CKT 98% 1%
Vero 99% 100%

 

Contaminating bacteria, fungi, mycoplasma and cytopathic viruses
were not observed. Electron microscopic observations of CKT cells
revealed particles similar to type C viral particles budding through the
cell membrane (Figure 12). These were also observed outside of the cell
in association with the cell membrane (Figure 13). Sizes ranged from
90 nm to 130 nm in diameter.

No gross or histopathologic lesions were observed in dogs and

hamsters inoculated with CKT cells.

43

 

Figure 11. Growth of CKT cells in soft
agar. Notice the vesicle formation due to
fluid transport activity (arrow). Unstained;
X 192.

 

44

  

.-.. ;t_
.- f , .- 2‘ —| '§.
.‘ ' .h‘.'6 '
.. _._ X ' O" .
- -. ~ .- - :-
‘ “ .-.k ' 1,};
. .‘ . . _‘ .. . .
4 I

n; 4

 
    
      
   
   

’Ik‘.UL ‘

    

 

. ‘

 
 

‘1'ng “
‘vaarfi v-2

-. Lip‘ o 1“ «(Li

Figure 12. Electron micrograph showing a
C-type virus particle budding from the cytoplasmic
membrane (arrow). Uranyl acetate and lead
citrate; X 52,500.

 

Figure 13. Electron.micrograph showing C-type
virus particles outside the cytoplasmic membrane
(arrows). Uranyl acetate and lead citrate; X 55,500.

 

45
DISCUSSION

The "blebbing" effect observed in CKT cells has been described in
other cell lines, viz., normal canine kidney (MDCK) (9), human breast
carcinoma (24), and mouse mammary tumor (13). Bleb formation is
probably a result of fluid transport activity as previously suggested
(9) but does not reflect an organ specific function. Rather, it appears
to be a cellular function of secretory epithelia which would indicate
.that other glandular tissue such as thyroid, adrenal, pancreas, and
mucous membranes are potentially capable of forming blebs in vitro.

The CKT cells grew rapidly, had a high growth efficiency, and
were capable of growth in several mediums. However, they could not be
maintained for extended periods of time and usually detached from the
surface within 2 weeks following the formation of a monolayer. Cellu-
lar growth was adversely affected by postconfluence inhibition as
indicated by the low stationary density.

Chromosomes of CKT cells were markedly different from normal dog
cells. There was a reduction of chromosomes with a corresponding
increase in the number of metacentric and submetacentric chromosomes.’
This phenomenon has been described in canine venereal tumors (2,11,
12,16,26) and cell lines derived from tumors of dogs (1,15,17). It has
also been reported in other animal species, particularly those that
have a high number of acrocentric chromosomes per cell (15). A possible
mechanism to explain the increase in metacentric and submetacentric
chromosomes is centric fusion, a process whereby 2 acrocentric chromo—

somes fuse at the centromere to form a single chromosome (15).

O...

46

The viral susceptibility spectrum of CKT cells was limited in
that only 8 of 27 viruses were successfully propagated. As the results
indicate, the CKT cell line offers no advantage over presently available
cell lines in propagating these viruses. The effects observed with
Eastern equine encephalomyelitis virus and Western equine encephalo-
myelitis virus was possibly due to an abortive infection, i.e., no
virions or only incomplete virions were produced. The abnormally large
syncytia produced by pseudorabies virus indicates that the cell membranes
of infected CKT cells are easily fused. The transparent areas observed
within the membrane of detached polykaryocytes may have been due to a
damaged sodium-potassium pump as a result of membrane incorporated
viral antigens. The nonfunctional pump would result in a loss of Ki
and a corresponding uptake of Na+ and Cl- causing an influx of water.
This increase of water within the cell would result in swollen cells
with a transparent appearance.

Growth of CKT cells in soft agar suspension indicates tumorigenic
capabilities; virtually without exception, cells capable of growing in
soft agar produce neoplaSms upon inoculation into a suitable host (4).
The fact that CKT cells did not produce tumors in the present study
may have been due to several factors, e.g., the immunogenicity of CKT
cells and the immune responsiveness of the animals employed.

The virus-like particles associated with CKT cells were similar
to C-type oncornaviruses (27). Their presence did not result in any
observable adverse effect.

The long term cultivation and cultural characteristics of CKT

cells fulfill the criteria of an established cell line. The fact that

47
they were derived from cancerous tissue, have neoplastic properties,
and release C-type viral particles may make them useful for cancer
research. Physiologists may also find CKT cells useful as they exhibit

fluid transport activity, a cellular function of secretory epithelia.

l.

2.

48
LITERATURE CITED

Adams, E. W., L. P. Carter, and W. J. Sapp. 1968. Growth and
maintenance of the canine venereal tumor in continuous culture.
Cancer Res. 2§3753-757.

Barski, G., and Fr. Cornefert-Jensen. 1966. Cytogenetic study
of sticker venereal sarcoma in European dogs. J. Nat. Cancer
Inst.‘§1:787—797.

Cornwell, H. J. C., A. R. Weir, N. G. Wright, and H. Thompson.
1970. The susceptibility of a dog kidney cell line (MDCK) to
canine distemper, infectious canine hepatitis and canine herpes
viruses. Res. Vet. Sci. ll;580-582.

Discussion Group #14. 1973. Criteria of neoplastic transforma—
tion. Conference on carcinogenesis testing in the development
of new drugs. National Academy of Sciences, National Research
Council.

Guash, R., W. L. Hard, and F. Smith. 1966. Characterization of
an established line of canine kidney cells (MDCK). Proc. Soc.
Exp. Biol. Med. lggg931-935.

Greene, A. E., L. L. Coriell, and J. Charney. 1964. A rapid
cytotoxic antibody test to determine species of cell cultures.
J. Nat. Cancer Inst. Q2:779-786.

Kasza, L. 1964. Establishment and characterization of canine
thyroid adenocarcinoma and canine melanoma cell lines. Am. J.
Vet. Res. 2231178-1185.

Lai, S. S., A. W. Roberts, G. R. Carter, and G. Jersey. 1973.
Biological characteristics and viral susceptibility of a rat

embryonic skin cell line (RESI). Am. J. Vet. Res. §§;97-102.

10.

11.

12.

13.

14.

15.

16.

49

Leighton, J., L. W. Estes, S. Mansukhani, and Z. Brada. 1970.

A cell line derived from normal dog kidney (MDCK) exhibiting
qualities of papillary adenocarcinoma and of renal tubular
epithelium. Cancer 2§51022-1028.

Macpherson, I. 1969. Agar suspension culture for quantitation
of transformed cells. Habel and Selzman (eds.). Fundamental
techniques in virology. Academic Press. pp. 214-219.

Makino, S. 1963. Some epidemiologic aspects of venereal tumors
of dogs as revealed by chromosome and DNA studies. Ann. New
York Acad. Sci. 19§;1106-1122.

Makino, S., T. Sofuni, and S. Takayama. 1962. Cytological
studies of tumors. XXXIX. A further study of the chromosomes
in venereal tumors of the dog. The Nucleus 2;115-122.

McGrath, C. M., S. Nandi, and L. Young. 1972. Relationships
between organization of mammary tumor and the ability of tumor
cells to replicate mammary tumor virus and to recognize growth
inhibitory contact signal in vitro. J. Virol. 2:367-376.

Merchant, D. J., R. M. Kahn, and W. H. Murphy. 1964. Enumeration
of chromosomes. In Handbook of cell and organ culture. Burgess
Publishing Co. USA. pp. 198-200.

Miles, C. P., G. Moldavannu, D. G. Miller, and A. Moore. 1970.
Chromosome analysis of canine lymphosarcoma: two cases involving
probable centric fusion. Am. J. Vet. Res._§1:783-789.

Oshimura, M., M. Sasaki, and S. Makino. 1973. Chromosomal band-
ing patterns in primary and transplanted venereal tumors of the

dog. J. Nat. Cancer Inst. 2151197-1203.

17.

18.

19.

20.

21.

22.

23.

24.

50

Pakes, S. P., R. A. Griesemer, and L. Kasza. 1965. Chromosome
analysis of 2 canine tumor cell lines. Am. J. Vet. Res. 2g:
837-843.

Patil, S. R., S. Merrick, and H. A. Lubs. 1971. Identification
of each human chromosome with a modified giemsa stain. Science
.lZ§:821-822.

Roberts, A. W., and G. R. Carter. 1972. Cell culture procedures.
I§_Simplified and economical cell culture techniques for labora—
tories beginning clinical veterinary virology. Michigan State
Agricultural Experiment Station No. 5677. pp. 3-25.

Rosanoff, E. I. 1959. Continuous cultivation of malignant cells
from a canine testicular tumor. Proc. Soc. Exp. Biol. Med.
1995384-387.

Sapp, W. J., and E. W. Adams. 1970. C-type viral particles in
canine venereal tumor cell cultures. Am. J. Vet. Res. 2151321-
1323.

Shannon, J. E., and M. L. Macy (ed.). 1972. lfl_The American type
culture collection registry of animal cell lines. 2nd ed.
American Type Culture Collection, Rockville, Md.

Siegfried, L. M., and C. Olson. 1972. Characteristics of avian
transmissible lymphoid tumor cells maintained in culture. J.
Nat. Cancer Inst. 4§:791-795.

Soule, M. D., J. Vazquez, A. Long, S. Albert, and M. Brennan.
1973. A human cell line from a pleural effusion derived from

a breast carcinoma. J. Nat. Cancer Inst. 2131409-1415.

51

25. Stulberg, S., W. F. Simpson, and L. Berman. 1961. Species-
related antigens of mammalian cell strains as determined by
immunofluorescence. Proc. Soc. Exp. Biol. Med. 108:434-439.

26. Takayama, S. 1958. Existence of a stemecell lineage in an
infectious venereal tumor of the dog. Jap. J. Genetics fig;
56-64.

27. Yosida, T. H. 1968. Relationship between chromosomal alteration
and development of tumors in vivo and in vitro. 12_Cancer
cells in culture. H. Katsuta (ed.). University Park Press,

USA. pp. 171-194.