TRANSFORMATION INDUCED BY PR 8 INFLUENZA VIRUS IN PRIMARY CULTURES OF MOUSE KIDNEY AND BRONCHUS, AND PRODUCTION OF MALIGNANT KIDNEY TUMORS IN MICE ...
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
TRANSFORMATION INDUCED BY PR 8 INFLUENZA VIRUS
IN PRIMARY CULTURES OF MOUSE KIDNEY AND BRONCHUS,
AND PRODUCTION OF MALIGNANT KIDNEY TUMORS IN MICE
BY SUBCULTURES*
BY CECILIE LEUCHTENBERGER, RUDOLF LEUCHTENBERGER, THEODOR BRUNNER.
DOROTHY NORLIN, AND SYLVIA WEISS
DEPARTMENT OF CYTOCHEMISTRY,
SWISS INSTITUTE FOR EXPERIMENTAL CANCER RESEARCH, LAUSANNE
Communicated by Clarence C. Little, December 16, 1964
The present investigation was stimulated mainly by previous observations of
Kotin and Wisely' and of Leuchtenberger et al.,2-4 who exposed mice to a combina-
tion of subacute respiratory infections with influenza virus and inhalation of
aerosols of hydrocarbons, or to a combination of such infections with inhalation of
cigarette smoke, respectively. In view of the fact that both studies suggested that
influenza virus may be implicated as a cofactor in the development of bronchogenic
carcinoma, a further exploration of the role which influenza virus may play in
malignant transformation seemed of special interest. Tissue cultures provide a
much better model than the living animal for the evaluation of time sequential
cellular alterations during transformation. In this investigation the effect of in-
fluenza virus on cells was studied in long-term tissue cultures using correlated
cytological and microspectrographic cytochemical techniques.5' 6
Evidence will be presented that PR 8 influenza virus evokes not only trans-
formation of cells in cultures similar to that described by Shein and Enders7 and
by Shein et al.8 in cells from human kidney cultures infected with SV40 virus, but
also that inoculation of subcultures into the living animal results in the production
of malignant tumors.
Materials and Methods.-An inbred strain of mice, characterized by a hereditary recessive an-
terior pituitary hypoplasia (Snell's dwarf mice) was utilized.9 We selected this strain because it
afforded the opportunity to compare the response of cells to infection with influenza virus in tissue
cultures from litter mates, which are dwarfs, with that of normal mice.'0 This report will, how-
ever, be concerned only with the effect of influenza virus on cells from "normal mice" of this strain.
Kidneys from mice (9-25 days old) and bronchi2 from normal mice (37-70 days old), were tryp-
sinized and grown on coverslips in plastic Petri dishes in Eagle's growth medium, modified ac-
cording to Dulbecco and Freeman," containing 10% calf serum, penicillin, and streptomycin, and
placed in a CO2 incubator at 370C. In 11 experiments comprising 338 primary cultures of kidneys,
168 cultures 3-14 days of age were infected with PR 8 influenza virus in a dose of 10-3, and 170
cultures were kept as controls. In 3 experiments comprising 41 primary cultures of bronchi, 22
cultures 9-12 days of age were infected in the same manner, and 19 cultures were kept as controls.
Subcultures were prepared from the infected and control cultures. Primary as well as subcul-
tures were injected into newborn Snell's mice intraperitoneally and intrathoracically. The
PR 8 influenza virus was originally obtained from Dr. Lindenmann of the Department of Pub-
lic Health in Bern, Switzerland, and had undergone an undetermined number of egg passages.
The viral material used for the infection of kidney and bronchi cultures consisted of allantoic
fluid of eggs infected with a high dilution material (10-6) and had a hemagglutination titer of 2560.
Infected cultures on coverslips were harvested at periods from 6 hr to 78 days after infection when,
at each age, coverslips of control cultures of the same age were also removed simultaneously.
For the cytological examination, coverslips of living cultures, and coverslips after fixation for 3
min in 95% alcohol, followed by dehydration and standardized staining with H.E. and Giemsa,
were examined by phase and light microscopy, respectively. Control and infected cultures were
694
Downloaded by guest on January 25, 2021VOL. 53. 1965 MICROBIOLOGY: LEUCHTENBERGER ET AL. 695
always mounted on the same slide, and studied first without knowledge of whether or not the
culture was infected. For a relative comparison of intracellular RNA and DNA, acridine-orange
fluorescence12 and methyl-green-pyronin staining's were used, and for the quantitative DNA
determinations in individual cells Feulgen microspectrography was applied as previously de-
scribed.14 For the hemadsorption studies, infected cultures (24 hr, 72 hr, 7 days, and 20 days
after infection) and corresponding control cultures were washed 3 times with PBS, and washed
guinea pig red cells were added at a concentration of 0.5%. These cells were left on the tissue
culture cells for a few minutes with occasional rocking of the Petri dish. The red cell suspension
was then drained and the cultures were washed once carefully with PBS. Hemadsorption was ob-
served under low-power and high-power magnification after staining with Giemsa and Feulgen
Fast Green.'4
Results.-When primary cultures of kidneys from Snell's mice (9-25 days old)
were examined at different periods after infection with PR 8 influenza virus, specific
cytological and cytochemical features were observed as summarized in Table 1
and as described below. The cellular alterations were reproducible in all of 168 in-
fected cultures from 11 experiments and were not found in 170 corresponding non-
infected control cultures of the same age. Although the changes in the cultures
were not always confined to a special stage, but were sometimes overlapping (partic-
ularly between stages I and II and between stages II and III), for convenience of
description the sequential alterations progressing with time are divided into 3 main
stages.
Stage 1: An increasing number of cells exhibit a marked enlargement, 24-72 hr
after infection, in nucleolar and nuclear size, with production of inclusions, first in
TABLE 1
COMPARISON BETWEEN ESSENTIAL CYTOLOGICAL AND CYTOCHEMICAL FEATURES*
Type of Stage I Stage 2 Stage 3
Feature culture 24-72 hr 72-144 hr 144 hr-78 days
Striking inhibition of mitosis Infected + -
Control - -
Marked proliferation Infected - + + (focal)
Control + + +
Striking chromosomal alterations Infected - + +
Control - -
Enlargement of nuclear and nucleolar Infected +
size characteristic for influenza virus (or rare)
infection Control
Striking degeneration and death of cells Infected + + + (focal)
Control - -
Disorganized cell pattern of culture and Infected - - +
occurrence of giant bizarrely shaped (or rare)
cells Control
Striking intranuclear and intracytoplasmic Infected +
RNA increase characteristic for influ- (or rare)
enza virus infection (viral replication) Control -
Abnormally high DNA content in nuclei Infected - - +
Control -
Striking tendency to higher DNA values Infected - + +
in areas of proliferation Control -
DNA and RNA increase in accordance Infected - + +
with cell division Control + + +
* In 170 noninfected controls and in 168 Sne Il's mouse kidney cultures after various periods of infection with
PR 8 influenza virus.
Downloaded by guest on January 25, 2021696 MICROBIOLOGY: LEUCHTENBERGER ET AL. PROC. N. A. S.
the nucleus, then in the cytoplasm, accompanied by formation of large quantities
of RNA, first present in nucleoli, nuclei, and then in cytoplasm, while the DNA
quantity remains unchanged. These cells do not show any evidence of cell di-
vision but, on the contrary, disclose gradual cell degeneration and cell destruction,
as indicated by breaking up, margination of nuclear chromatin, and ballooning of
nuclei, with gradual loss of RNA and finally of DNA from cells. This sequence of
cytological and cytochemical events is essentially the same as described after in
vivo infections with PR 8 influenza virus.4e 6 These sequential changes were never
found in control cultures.
Stage II: From 72 to 144 hr after infection, areas of cell necrosis become more
prominent, but at the same time foci of pronounced cellular proliferation are found,
which frequently seem to be heaping up and display a crisscross appearance.
Such proliferating cells do not reveal any of the morphological and nucleic acid
alterations described in stage I. They resemble those found in areas of rapid pro-
liferation in noninfected control cultures, that is, both exhibit mitosis and carry in-
tracellular DNA and RNA augmentations in accordance with the process of cell
division. However, the tendency to crisscross growth seems to be somewhat more
frequent in the infected cultures. Furthermore, as can be seen from Figure 1, the
intracellular DNA content in areas of proliferation of infected cultures is, in general,
higher than in cells of similar areas of control cultures. This difference in the DNA
content in areas of proliferation not only holds true for the primary cultures, but
also for the subcultures.
Stage III: From 144 hr up to the termination of the experiment (the longest
being, 78 days), striking cellular alterations which have never been seen in the
corresponding control cultures can be observed in the infected cultures. In con-
trast to the controls, which form now a rather even sheet of relatively uniform
cells (Fig. 2), the infected cultures show a disorganized pattern, such as marked
disparity of size of cells, of nuclei, and of nucleoli and their number. There is also
disruption of cell boundaries, and ragged appearance of cytoplasm with frequent
vacuolization.
Within areas of rarefication, where signs of preceding cell degeneration can
still be seen, a number of extraordinarily large cells with bizarrely shaped giant
nuclei, frequently disclosing budding and formation of micronuclei, and highly ab-
normal distribution of clumped large quantities of chromatin are noted (Figs. 3a,
3b). Some of these cells exhibit abnormal prophases and metaphases, tripolar
spindles, and marked alterations of chromosomes, such as stickiness, lagging, and
feathering. DNA analyses of these abnormal cells by microspectrography yield
values approximately 90 times, while in control cultures the largest nuclei, which
were rather rare and of normal shape, yield values up to 16 times the amount of a
diploid nucleus.
Essentially the same findings as those described for kidney cultures after infec-
tion with PR 8 influenza virus were obtained in infected cultures of bronchi from
normal Snell's mice. However, since control cultures of bronchi from these adult
mice revealed a much more variable morphological and DNA pattern of the cells,
an assessment of the changes induced by the infection with influenza virus of bron-
chial epithelium in cultures presents difficulties.
In view of the fact that abnormal cells in the infected cultures (Figs. 3a, 3b)
Downloaded by guest on January 25, 2021VOL. 53, 1965
1.
60
42
4
N =,
2 CNA
2
2
N 7 '7
_
.1!:ilkW1]|~Ili"[!1.
MICROBIOLOGY: LEUCHTENBERGER ET AL.
C- NT.R A_
4
4
6
6
AMOUNT OF DNA IN ARBITRARY UNIT S
N * NUMBER OF NUCLEI
8
12 !6
FIG. 1.-Comparison of DNA content in individual nuclei from
proliferating areas of control and PR 8 influenza virus-infected
Snell's kidney cultures.
resemble closely those described by Shein and Enders from human kidney cultures
infected with SV40 virus,' 8 and also exhibit characteristics usually associated
with tumor cells, primary cultures and subcultures containing these grotesque
cells were injected intraperitoneally and intrathoracically into newborn Snell's
mice. While the majority of the mice injected with primary cultures are still alive
and so far without macroscopic evidence of tumor (the longest period after in-
jection being 9 months), a few mice were killed within the first two weeks for the
...... .. ..,...
18
,, .L01UaIL
20-26
697
A~~~~~~~~~~~~~K
.14
~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 4
.4~~~~~~~~~~~~~~.
FIG. 2 -Ten-day old culture of Snell's kidneys' Noninfected culture, Feulgen reaction, approx
X 375. Note even appearance of culture and normal shape of nuclei of varying sizes.
Downloaded by guest on January 25, 2021698
*
..........
........
;-
' ~
....VOL. 53, 1965 MICROBIOLOGY: LEUCHTENBERGER ET AL. 699
FIG. 4.-Section of small bronchus of 11-day-old mouse. Intrapulmonary injection, at birth,
of mouse kidne cell culture, 7 days previously infected with PR 8 influenza virus (see Fig. 3a).
H.E. X 375. Note several rows of proliferating, focally irregularly arranged epithelial cells with
abundant mito-ses. Beginning stratification and squamous cell metaplasia of lining epithelium.
In the outer part of bronchial lumen, mixed iiiflammatory and few desquamated epithelial cells.
in Figure 5. This tumor occurred 3 months after intrathoracic injection of a
2nd passage derived from an explant of bronchus infected with PR 8 influenza
virus. Both kidneys were involved. The left kidney was greatly enlarged and
consisted to over 90 per cent of a solid tumor mass which had infiltrated capsule
and peritoneum; the right one was of normal size and disclosed tumor in the renal
medulla infiltrating the renal pelvis. Microscopically, masses of spindle cell sar-
coma were present.
Discussion.-There are two findings in the present study which deserve special
consideration:
(1) Cells from Snell's mouse kidney cultures respond in essentially the same
manner to PR 8 influenza virus as do cells from Snell's mouse bronchi cultures and
cells from CF1 mouse bronchi after in vivo infection with PR 8 influenza virus.
Downloaded by guest on January 25, 2021700 MICROBIOLOGY: LEUCHTEANBERGER ET AL. PROC. N. A. S.
.Wg5
FIG. 5.-Section of kidney tumor of 2-month-old mouse. Intrathoracic injection, at birth, of
2nd passage derived from bronchus previously infected with PR 8 influenza virus. H. E approx.
X 375. Note interlacing bundles of highly atypical cells with mitosis characteristic of poorly dif-
ferentiated spindle cell sarcoma.
In all instances the influenza virus evokes two opposite effects which follow each
other within different cells of the same host tissue, namely, virus-type specific
nucleic acid (RNA) replication resulting in cell death, followed by cell-specific DNA
and RNA replication resulting in cell proliferation and eventual transformation.
The observation that kidney cultures were also suitable for the exploration of the
biological effect of PR 8 influenza virus proved to be of advantage, not only because
dissection and preparation of bronchi from mice for tissue cultures are rather
difficult, but especially in view of the fact that, in contrast to control cultures of
kidneys, control cultures of bronchi sometimes revealed cellular abnormalities.
(2) All cultures of mouse kidneys and bronchi infected with PR 8 influenza
virus revealed giant, bizarrely shaped cells exhibiting abnormal mitosis and high
DNA content. These cells are not only strikingly similar, in regard to morphology,
to those observed after infection of human kidney cultures with SV4O virus,7s 8 but
subcultures, when injected into newborn mice, also produce malignant tumors as
do hamster cells infected with SV4O virus.'15, 16
It thus appears that PR 8 influenza virus is another respiratory "infectious"
virus which under certain conditions is capable of eliciting malignant transforma-
tion of cells as has been demonstrated for adenovirus types 12 anid 18 by Trentin
et al.'7 This findingg does not only give further support to the concept that in-
fluenza virus may be iniplicated as a cofactor inl the development of bronchogenic
carcinoma,'- but it imposes caution on the strict separation between "tumor"
viruses and "infectious" viruses.6
Immunological studies are under way to explore the interrelation between PR 8
Downloaded by guest on January 25, 2021VOL. 53, 1965 MICROBIOLOGY: LEUCHTENBERGER ET AL. 701
influenza virus, morphological and cytochemical transformation of cells in tissue
cultures, and the malignant property of these cells.
Summary.-Cultures from kidney and bronchus of Snell's mice, when infected
with PR 8 influenza virus, showed essentially the same sequence of cytological and
cytochemical alterations as those observed in bronchi and lungs of CF1 mice after
in vivo infection with PR 8 influenza virus. In addition, there oceurred in the in-
fected tissue cultures giant bizarrely shaped cells resembling closely those de-
scribed by Shein and Enders for cells from human kidney cultures infected with
SV40 virus. Intraperitoneal and intrathoracic injection of newborn mice with
subcultures derived from PR 8 infected explants of kidney and bronchus resulted
in production of malignant kidney tumors.
The authors wish to express their great gratitude to Dr. John F. Enders, Department of Bac-
teriology and Immunology, Harvard Medical School, Boston, Mass., for his interest and valuable
advice in this work. The authors are also indebted to Dr. Schultz-Larsen, Institute of Human
Genetics in Copenhagen, for sending Snell's mice.
* This study was supported in part by research grants of the Council for Tobacco Research,
U.S.A., and of the Ciba, Basel, Switzerland.
I Kotin, P., and D. V. Wisely, in Progress in Experimental Tumor Research, ed. F. Homburger
(Basel and New York: Karger, 1963), vol. 3, p. 186.
2 Leuchtenberger, C., R. Leuchtenberger, and P. F. Doolin, Cancer, 11, 490 (1958).
3Leuchtenberger, C., R. Leuchtenberger, W. Zebrun, and P. Shaffer, Cancer, 13, 721 (1960).
4Leuchtenberger, C., R. Leuchtenberger, F. Ruch, K. Tanaka, and T. Tanaka, Cancer Res.,
23, 555 (1963).
5 Leuchtenberger, C., and R. Leuchtenberger, Intern. Rev. Cytol., 14, 281 (1963).
6 Leuchtenberger, C., Bibl. Microbiol. (Basel and New York: Karger, 1964), Fasc. 4, p. 18.
Shein, H. M., and J. F. Enders, these PROCEEDINGS, 48, 1164 (1962).
8 Shein, H. M., J. F. Enders, L. Palmer, and E. Grogan, Proc. Soc. Exptl. Biol. Med., 115, 618
(1964).
9 Kemp, T., Acta Pathol. Microbiol. Scand., Suppl. 16, 189 (1933).
10 Leuchtenberger, C., H. Fr. Helweg-Larsen, and L. Murmanis, Lab. Invest., 3, 245 (1954).
11 Dulbecco, R., and G. Freeman, Virology, 8, 396 (1959).
12 Niven, J. S. F., "The cytopathology of virusinfection," Ann. N. Y. Acad. Sci., 81,84 (1959).
13 Leuchtenberger, C., Chromosoma, 3, 449 (1950).
14 Leuchtenberger, C., in General Cytochemical Methods, ed. J. F. Danielli (New York: Academic
Press, 1958), p. 219.
1" Rabson, A. S., and R. L. Kirschstein, Proc. Soc. Exptl. Biol. Med., 111, 323 (1962).
16 Shein, H. M., J. F. Enders, Y. D. Levinthal, and A. E. Burket, these PROCEEDINGS, 49, 28
(1963).
17 Trentin, Y. Y., Y. Yabe, and G. Taylor, Science, 137,835 (1962).
Downloaded by guest on January 25, 2021You can also read
