Human Cells Transformed In Vitro by Human Cytomegalovirus: Tumorigenicity in Athymic Nude Mice 1 , 2 L. Geder,
3
J. Kreider, 4 and F. Rapp 3, 5
ABSTRACT-Athymic nude mice were inoculated with human embryo lung cells transformed in vitro by human cytomegalovirus (CMV). Of the inoculated animals, 62% developed tumors after an average latent period of 19 days. The tumors were composed of small, polygonal cells with large nuclei and scanty cytoplasm embedded in an abundant collagenous matrix. The cells were poorly differentiated but may have been of epithelial origin. Adjacent structures were rarely invaded. CMV-related intracellular and membrane antigens were detected by indirect and anticomplement immunofluorescence techniques in cells cultured in vitro from the tumors.-J Natl Cancer Inst 58: 1003-1009, 1977.
tive for CMV-related antigens during in vitro passages. The tumors grew progressively in the nude mice. Although poorly differentiated, the tumor cells may have been of epithelial origin. Control uninfected HEL cells or latently infected human prostatic fibroblasts were nononcogenic in the nude mice. The results indicate that the oncogenicity of the CMV-Mj-HEL-2 cells is the consequence of in vitro transformation of the HEL cells by CMV. MATERIALS AND METHODS
A primary, but often unrealized, goal of the study of virus-transforming potential is transformation of human cells and subsequent demonstration of the oncogenic capacity of the transformants. The availability of athymic nude mice has made such experiments feasible, and thus observations of in vivo growth and antigenic variations of transformed human cells become possible. Previous studies have shown that irradiated human CMV retains ability to transform hamster embryo fibroblasts (l) and that the transformed cells are oncogenic in newborn hamsters. Recently, we reported that a human CMV of prostatic origin established a long-term persistent infection in HEL cells in vitro (2). After a number of cell culture passages, foci of short fibroblastoid and epithelioid cells developed, and two cell lines (designated CMV-Mj-HEL-l and CMV-Mj-HEL-2) were obtained in which foci of infection could no longer be demonstrated and from which virus could not be rescued. The cells ceased to demonstrate contact inhibition. CMV-specific antigens were demonstrated in both cell lines by the indirect IF technique. Microcytotoxicity tests established that CMV-Mj-HEL-l and CMV-MjHEL-2 cells, but not control HEL cells, share a membrane antigen(s) with hamster cells transformed by CMV. Initial studies indicated that the CMV-Mj-HEL-l cells were oncogenic in athymic nude mice and that cells cultured in vitro from the tumor had CMV-related antigens. However, karyotypic analysis could not exclude the possibility that the HEL cells became contaminated with an established human bladder tumor cell line during the period of persistent infection with CMV and that the bladder cells were subsequently transformed with CMV. The original bladder cells were oncogenic for the athymic nude mice, and thus the oncogenicity of the CMV-Mj-HEL-l cells could be an independent phenomenon and not the consequence of CMV transformation (3). The HEL origin of the second transformed cell line (CMV-Mj-HEL-2) has been confirmed by karyotype analysis. This report confirms the oncogenicity of the CMVtransformed human cells in athymic nude mice and provides evidence that passage in vivo does not lead to loss of the CMV-specific antigens in the tumor line. Cell cultures established from these tumors remained posiVOL. 58, NO.4, APRIL 1977 Downloaded from https://academic.oup.com/jnci/article-abstract/58/4/1003/908462 by University of California, Santa Barbara user on 04 March 2018
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Cells.-CMV-Mj-P cells originating from human prostate tissue that apparently had been infected in vivo with CMV were grown in Ham's medium supplemented with 20% FCS, 0.075% sodium bicarbonate, 100 IV penicillin/ ml, and 100 JLg streptomycin/ml. The cells grew in vitro to passage levels well above those routinely attained by normal cells. After a number of passages, virus was no longer rescuable, although CMV-specific antigens and nucleic acid were continually detected in the cells (2). Cells of the CMV-Mj-HEL-l and CMV-Mj-HEL-2 lines were maintained in Ham's medium with the same supplements. Animals.-Weanling athymic homozygous (nu/nu) nude mice (NIH Swiss Webster, sixth backcross generation) were obtained from Life Sciences, St. Petersburg, Florida. Inoculation of athymic nude mice with CMV-transformed HEL cells.-Cell sheets were trypsinized with a mixture of 0.1 % trypsin and 0.02% Versene and washed by centrifugation three times in Ham's medium with 0.075% sodium bicarbonate, 100 IV penicillin/ml, and 100 JLg streptomycin/ml. The cells were resuspended in the same medium and counted. Cells were inoculated sc CMV = cytomegalovirus; HEL = human embryo lung; IF = immunofluorescence; FCS = fetal calf serum; HSV-l = herpes simplex virus type 1; HSV-2 = herpes simplex virus type 2; PARA = particle aiding replication of adenovirus; SV40 = simian virus 40; ACIF = anticomplement immunofluorescence; FITC = fluorescein isothiocyanate; IUDR = 5-iodo-2'-deoxyuridine; DMSO = dimethyl sulfoxide. 1 Received June 7, 1976; accepted September 27, 1976. 2 Supported by Public Health Service (PHS) contract NOI CP53516 from the Virus Cancer Program, Division of Cancer Cause and Prevention, National Cancer Institute (NCI), and by PHS grant CA16365 from the NCI. 3 Department of Microbiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, Hershey, Pa. 17033. 4 Departments of Pathology and Microbiology, The Milton S. Hershey Medical Center. 5 We thank Dr. J. Gruber, Office of Program Resources and Logistics, Viral Oncology Program, Division of Cancer Cause and Prevention, NCI, Bethesda, Md., for supplies of nude mice; Dr. F. O'Neill, Department of Microbiology and Pathology, University of Utah Medical Center, Salt Lake City, Utah, for karyotype analysis; and Judith Blom and Julie Miskimen, Department of Microbiology, The Milton S. Hershey Medical Center, for technical assistance. ABBREVIATIONS USED:
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into the middle of the back of newborn and weanling (2to 3-wk-old) nude mice. Autopsy and histologic examination. -Tumor-bearing animals were killed with chloroform. Tissue was removed, fixed in neutral buffered formalin, cut into 5-/L paraffin sections, and stained with hematoxylin and eosin. Preparation of tumor cell cultures.-Half of the tumors were washed three times in phosphate-buffered saline, minced into 1- to 2-mm pieces, and transferred into plastic tissue culture flasks (surface area, 28 em") in Ham's medium with 20% FCS, 0.075% sodium bicarbonate, 100 IU penicillin/ml, and 100 /Lg streptomycin/mi. The fragments attached to the surface, and the cells spread and formed a confluent cell sheet within 1 week. The tumor cell cultures were normally maintained by dividing the cells from one flask into two every second day. Stained cover slip preparations. -Tumor cells grown on cover slips were fixed in methanol for 2 minutes at room temperature and were then stained with May-Griinwald-Giemsa. Immune sera.-Human antisera to CMV were obtained from hospital patients. Immune sera "W" and "T" had indirect IF antibody titers to CMV-infected HEL cells of 1:128 and 1:256, respectively. Serum Wand serum T had titers of 1:64 to HSV-1-infected HEL cells and 1:32 to HSV-2-infected HEL cells. The HSV-2-specific hamster serum had an indirect IF antibody titer to HSV-2infected HEL cells of 1:32, and sera from PARA (defective SV40)-adenovirus 7 tumor-bearing hamster reacted with homologous tumor cells at a titer of 1:8 in the indirect IF test. All immune sera used in the indirect IF and ACIF tests were adsorbed with 108 HEL cells/rnl of undiluted serum for 1 hour at 37° C and then overnight at 4° C before use. One sample of immune serum W was adsorbed with CMV-infected HEL cells (108 cells/0.5 ml of undiluted immune serum; multiplicity of infection=0.3 plaque-forming unit/cell). Serologic tests.-Standard procedures of the indirect IF test were used for the detection of CMV-specific antigens in the transformed cells (2). Goat anti-human IgG conjugated to FITC was purchased from Cappel Laboratories, Downingtown, Pennsylvania. In the ACIF test, cover slip cultures were fixed with a mixture of equal parts of methanol and acetone for 2.5 minutes, covered
with inactivated anti-CMV serum, and incubated at 37° C for 45 minutes. After repeated washings, 2-4 U of complement was added for 45 minutes at 37° C. CMV antibody-negative human serum served as the complement source. The cells were washed again with phosphate-buffered saline and covered with goat antihuman complement serum labeled with FITC for 45 minutes at 37° C. After final washings, the cells were mounted on slides in a mixture of 1 part phosphate-buffered saline and 9 parts glycerol. Virus rescue experiments.-Several different procedures were used to rescue an infectious agent from the CMV tumor cells. Cocultivation experiments were performed by seeding 2 x 106 HEL cells into 30-ml plastic flasks. The following day, 2 x 106 tumor cells were added. A confluent monolayer of the mixed culture developed within 1-2 days. In some cocultivation experiments, 100 /Lg IUDR/ml (Calbiochem, San Diego, Calif.) was added to the culture for 3 days. HEL cells were also inoculated with medium from tumor cell cultures or extracts of the cells disrupted by sonication. Tumor cells were also subjected to UV irradiation for 4 minutes at a distance of200 mm (42 ergs/sec/rum") and then seeded into 30-ml tissue culture flasks. The irradiated cells were sometimes cocultured with HEL cells. Treatment with cytochalasin B. -Cells (0.5 x 105 ) were grown on cover slips in 60-mm petri dishes containing normal medium. When the cells were attached to the cover slips, the normal medium was replaced with medium containing 1-3 /Lgcytochalasin B/ml. The cultures were incubated in the medium for 7 days and were refed twice during this period. The cells were fixed in 3 parts of methanol and 1 part glacial acetic acid; they were then stained with May-Griinwald-Giemsa and assayed for multinucleation. Between 250 and 300 consecutive cells were scored and the data recorded. The controls received DMSO, the cytochalasin B solvent, at 0.075%. RESULTS Tumors in Athymic Nude Mice Inoculated With CMV-Transformed Cells (CMV-MJ-HEL-2)
As shown in table 1, 33 of 53 athymic nude mice inoculated 2-4 weeks after birth with different numbers of CMV-Mj-HEL-2 cells developed tumors after a mean latent period of 19 days. All of the nude mice inoculated
TABLE I.-Development of tumors in athymic nude mice inoculated with CMV-transformed HEL cells
a
Group
Age at inoculation, wk
Cell line used
Dose of cells
Passage No.
1 2 3 4 5 6 7 8 9 10
3 2 3 2 3 3 4 4 2 3
CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-P HEL
4xl0 7 4x10 7 4x10 7 4x10 7 107 5x10· 10· 5x105 4x10 7 2x10 7
6 8 12 13 62 62 79 79 11-75 10
Number with tumor/No. inoculated 8/14 5/5 6/14 3/6 5/5 3/3 2/3 1/3 0/85 0/10
Mean latent period, days 20 19 14 17 14 18 20 13
Histology a PDT PDT PDT PDT PDT PDT PDT PDT G
PDT=Poorly differentiated tumor cells; G=granuloma.
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with 5x 106 , 107 , 2x 107 , or 4x 107 CMV-Mj-HEL-2 cells developed tumors. Only 2 of 3 nude mice inoculated with 106 transformed cells and 1 of 3 nude mice inoculated with 5 x 105 transformed cells developed tumors. No tumors developed in animals inoculated with 105 cells during their lifetime of about 8 weeks. The average size of the tumors at 3 weeks was approximately 16x 14 mm (figs. 1,2). Tumors were rather soft and occasionally attached to underlying normal tissues. In tumors removed 6-8 weeks after cell injection, central necrotic areas were common. The tumors consisted of poorly differentiated cells, possibly of epithelial origin. The cells were small and polygonal, with large nuclei and scanty, clear cytoplasm. The cells were often embedded in an abundant collagenous matrix (fig. 3). Adjacent structures, e.g., panniculus carnosus, were rarely invaded. No tumors were observed in 10 nude mice inoculated with control HEL cells, nor did tumors develop in 85 nude mice inoculated with CMV-Mj-P cells. However, 19 nude mice of this group developed granulomas after a mean latent period of 8 days. The granulomas consisted of necrotic material, presumably derived from the inoculum, which was surrounded by intensive infiltrates of neutrophils and granulation tissue composed of fibroblasts, capillary buds, and mononuclear leukocytes. Tumor Cell Cultures
The tumor cells cultured in vitro were small and epithelioid (fig. 4). Multilayered growth developed when the cell cultures were maintained without passage after development of a confluent cell sheet. The overgrowing cells were of rounded morphology and tended to detach and float in the medium. Concentration densities of approximately 106 cells/em! were reached with transformed cells, while normal HEL cells grew to 4x 105 cells/em", Karyotype and Effect of Treatment With Cytochalasin B
The parental CMV-carrier HEL cells (CMV-Mj-P) contained 46 chromosomes, had no numerical or strucTABLE
tural markers, were female, and responded with controlled nuclear division to treatment with cytochalasin B. The CMV-Mj-HEL-2-transformed and CMV-MjHEL-2,T-l tumor cells contained 47-48 chromosomes, had no numerical but some structural markers, belonged to the same sex, but developed semi-uncontrolled nuclear division after treatment with cytochalasin B (table 2). Virus Rescue Studies
No infectious virus was recovered from the tumor cells by inoculation of cell extracts onto HEL cultures or by cocultivation with HEL cells. Irradiation with UV light (4 min at a distance of200 mm, 42 ergs/sec/rnm") or pretreatment with 100 /-Lg IUDR/ml in medium for 3 days also failed to induce infectious virus. Serologic Detection of CMV-Specific Antigens
CMV-specific membrane antigens were detected in the tumor cells in different in vitro passages by indirect IF with the use of two different human convalescent sera (fig. 5, table 3). Nuclear fluorescence was observed in most cells when the ACIF technique was employed (fig. 6). The antisera did not react with normal HEL cells. CMV-immune serum was adsorbed with CMVinfected HEL cells (l08 cells/ml immune serum) for 1 hour at 37° C and then overnight at 4° C. This adsorption largely reduced the reactivity of the immune serum for the CMV tumor cell membrane. Adsorption with normal HEL cells did not affect reactivity. HSV-2-immune sera and sera from PARA(defective SV40)-adenovirus 7 tumor-bearing hamsters did not react with CMV tumor cells (table 2). DISCUSSION
We reported previously (2) that human prostate cells, which apparently were infected in vivo with CMV, initially released the virus in cell culture. During subsequent passage of the cells, release of virus could no longer be detected. However, CMV-specific membrane antigens were still demonstrable via several imrnuno-
2.-ehromosome constitution ofCMV-carrier and CMV-transformed cells Numerical markers
Cell line
Chromosome No.
CMV-Mj_Pb CMV-Mj-HEL-2 c
47-48
CMV-Mj-HEL-2,T-ld
47-48
6 G-group chromosomes
7 D-group
chromosomes
5 B-group chromosomes
Structural markers
Sex
Comments"
CND SUND
46
1 minute 3 small 1 metacentric 1 funny D 1 minute 3 small 1 metacentric 1 funny D
SUND
e CND=controlled nuclear division after treatment with cytochalasin B; SUND=semi-uncontrolled nuclear division after treatment with cytochalasin B. b CMV-Mj carrier HEL cell line, source of CMV-transformed cells. c CMV-transformed human cells. Transformed cells developed at passage 3 after inoculation with CMV. d Tumor cell culture. Tumors were induced in weanling athymic nude mice with CMV-Mj-HEL-2 cells.
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3.-Presence ofCMV-associated antigens on the surfaces of tumor cells from athymic nude mice In vitro passage
Cells CMV-Mj-HEL-2,T-1
f
CMV-Mj-HEL-2,T-2f CMV-Mj-HEL-2,T-3f HEL control HEL plus HSV-2 SV40-transformed
10 11 23 28 71 4
11 12 7 9
Specificity of antisera a Origin of cells Group 4"
Group 5" Group 7" Hamster tumor
CMVb
CMV-a c
41-49 52 41 40 40 45 50 35 45
2-5 5 ND ND ND ND ND ND ND
+
+
HSV-2 d
+
SV40e
+h (nuclear)
Indirect IF. Human convalescent serum adsorbed with HEL cells. Numbers are percent of cells with fluorescence. C Human convalescent serum adsorbed with CMV-infected HEL cells. Numbers are percent of cells with fluorescence. ND=not done. d Hamster serum adsorbed with HEL cells. e Serum from hamster bearing PARA(defective SV40)-adenovirus 7 tumor, adsorbed with HEL cells. f Tumor cell culture. Tumors were induced in weanling athymic nude mice with CMV-transformed human cells, designated CMV-Mj-HEL-2. o See table 1. h Fixed preparation. a b
logic assays, and hybridization studies with CMV -cornplementary RNA and cell DNA indicated that an average of 12 virus genome equivalents were present per cell. The CMV-Mj-P cells lost contact inhibition by passage 23 and continued to grow, reaching passage 81 some 40 weeks after their establishment in culture. Taken together, these observations suggest that the CMV-Mj-P cells were transformed by CMV. We have not, however, been able to establish a cell line beyond passage 86, and no tumors have been induced with these cells in athymic nude mice. Lang et al. (4) reported that CMV-infected human cells may acquire a morphology reminiscent of transformed cells prior to lysis. Thus the loss of contact inhibition, normally an indicator of transformation, may be misleading. However, it is also known that human transformed cells do not invariably induce tumors in nude mice (5). In subsequent experiments, HEL cells which were infected with a low multiplicity of CMV-Mj virions developed a long-term persistent infection with continuous development of CMV-specific nuclear and cytoplasmic antigens and release of infectious virus. During passage, some of these cultures went into crisis; shortly thereafter, foci of short fibroblastoid and epithelioid cells emerged and a confluent monolayer formed within 1 week. During subsequent passage of these cells, release of virus could no longer be detected. However, CMV-specific antigens continued to be synthesized. The CMV-Mj-HEL-l and CMV-Mj-HEL-2 cells lost contact inhibition and continued to grow, reaching passage 150 some 50 weeks after the first foci of transformed cells developed in the virus-carrier culture. The CMV-MjHEL-2 cells were used in these experiments because karyotypic analysis confirmed their HEL origin. Our goal was to detect direct correlation between the state of transformation by CMV and the oncogenic potential of the transformed cells in the athymic nude mice. We also
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wished to determine whether the CMV markers of the transformed cells survive the in vivo passage followed by numerous in vitro passages. In contrast to control HEL and CMV-Mj-P cells, the CMV-Mj-HEL-2 cells produced tumors when inoculated into a number of athymic nude mice. Thus the acquisition of oncogenic potential is a direct consequence of transformation by the virus. A most compelling argument against chronic infections may be made, because cells cultured in vitro from the tumor contained intracellular and membrane CMV-specific antigens detected by indirect IF as late as 71 in vitro passages after isolation. Thus passage in vivo, followed by numerous in vitro passages, did not lead to loss of the CMV-specific antigens. The tumor cells grew in vitro to high concentration densities. Previous publications have shown that cells transformed by SV40 and polyoma virus and those derived from neoplastic tissues become highly multinucleated following cytochalasin B treatment (6, 7-12). Conversely, normal and RNA virus-transformed cells become only binucleated (6, 9-12). Thus the uncontrolled nuclear division of our CMV tumor cells following cytochalasin B treatment is an indirect biologic evidence of transformation by a DNA virus. Interestingly, DNA analogue treatment was not required to obtain positive results in IF tests. The finding that CMV-immune sera reacted with the membranes of these cells and that this reactivity was specifically reduced when the immune sera were adsorbed with CMVinfected HEL cells confirms the CMV specificity of these antigens. When transplanted into athymic nude mice, human embryo fibroblasts transformed by a prostatic isolate of CMV have induced progressively growing tumors. Cell passage in vivo did not result in loss of antigens. The demonstrated capacity of CMV to transform human VOL. 58, NO.4, APRIL 1977
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cells and the demonstration of CMV antigens in human prostate cancer cells (13) provide further evidence of the possible oncogenic potential of this virus in its natural host. REFERENCES (1) ALBRECHT T, RAPp F: Malignant transformation of hamster em-
(2)
(3)
(4)
(5)
bryo fibroblasts following exposure to ultraviolet-irradiated human cytomegalovirus. Virology 55:53-61, 1973 RAPp F, GEDER L, MURAsKo D, et al: Long-term persistence of cytomegalovirus genome in cultured human cells of prostatic origin. J Virol 16:982-990, 1975 GEDER L, LAUSCH R, O'NEILL F, et al: Oncogenic transformation of human embryo lung cells by human cytomegalovirus. Science 192:1134-1137, 1976 LANG DJ, MONTAGNIER L, LATARJET R: Growth in agarose of human cells infected with cytomegalovirus. J Virol 14:327-332, 1974 ROBERTS DW, WEIDANZ DP: Tumor growth in congenitally athymic (nude) mice induced by the injection of MA-160 cells. Annual Meeting of the American Society of Microbiologists, abstract E73:73, 1975
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(6) WRIGHT WE, HAYFLICK L: Formation of anucleate and multinucleate cells in normal and SV40 transformed WI-38 by cyrochalasin B. Exp Cell Res 74: 187-194, 1972 (7) O'NEILL FJ, RAPp F: Premature chromosome condensation ir hamster cells treated with cytochalasin B. Exp Cell Res 70: 226229, 1971 (8) O'NEILL FJ: Chromosome pulverization in cultured normal anc neoplastic cells treated with cytochalasin B. J Nat! Cancer Ins! 49: 1733-1738, 1972 (9) - - - : Control of nuclear division in normal but not in neoplastic mouse cells. Cancer Res 34:1070-1073,1974 (10) - - - : Limitation of nuclear division by protease inhibitors ir cytochalasin-B-treated tumor cells. J Nat! Cancer Inst 52:653657,1974 (11) KELLY F, SAMBROOK J: Differential effect of cytochalasin B or normal and transformed mouse cells. Nature [New Biol 242:217-219, 1973 (12) HIRANO A, KURIMURA T: Virally transformed cells and cytochal asin B. I. The effect of cytochalasin B on cytokinesis, karyoki nesis and DNA synthesis in cells. Exp Cell Res 89:111-120, 197~ (13) GEDER L, SANFORD EJ, ROHNER TJ, et al: Cytomegalovirus anc cancer of the prostate: In vitro transformation of human cells Cancer Ther Rep. In press
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FIGURE FIGURE FIGURE
l.-Athymic nude mouse 24 days after first sign of development of tumor following inoculation with 4x 107 CMV-Mj-HEL-2 cells. 2.-Tumor of athymic nude mouse (fig. 1) induced by CMV-Mj-HEL-2 cells. 3.-Photomicrograph of tumor induced by CMV-Mj-HEL-2 cells in athymic nude mouse. Hematoxylin and eosin. x 500
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FIGURE 4.-Photomicrograph of in vitro culture of CMV-Mj-HEL-2,T-I tumor cells of athymic nude mouse. x 500. FIGURE 5.-Photomicrograph of membrane fluorescence detected on CMV-Mj-HEL-2,T-I tumor cells (passage 15) after reaction with CMVspecific human convalescent serum and FITC-conjugated goat anti-human IgG. x 1,250. FIGURE 6.-Photomicrograph of nuclear fluorescence detected in CMV-Mj-HEL-2,T-I tumor cells (passage 22). CMV-specific human convalescent serum was used with human complement and FITC-conjugated anti-human complement goat serum in the ACIF test. x 1,250.
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3
J. Kreider, 4 and F. Rapp 3, 5
ABSTRACT-Athymic nude mice were inoculated with human embryo lung cells transformed in vitro by human cytomegalovirus (CMV). Of the inoculated animals, 62% developed tumors after an average latent period of 19 days. The tumors were composed of small, polygonal cells with large nuclei and scanty cytoplasm embedded in an abundant collagenous matrix. The cells were poorly differentiated but may have been of epithelial origin. Adjacent structures were rarely invaded. CMV-related intracellular and membrane antigens were detected by indirect and anticomplement immunofluorescence techniques in cells cultured in vitro from the tumors.-J Natl Cancer Inst 58: 1003-1009, 1977.
tive for CMV-related antigens during in vitro passages. The tumors grew progressively in the nude mice. Although poorly differentiated, the tumor cells may have been of epithelial origin. Control uninfected HEL cells or latently infected human prostatic fibroblasts were nononcogenic in the nude mice. The results indicate that the oncogenicity of the CMV-Mj-HEL-2 cells is the consequence of in vitro transformation of the HEL cells by CMV. MATERIALS AND METHODS
A primary, but often unrealized, goal of the study of virus-transforming potential is transformation of human cells and subsequent demonstration of the oncogenic capacity of the transformants. The availability of athymic nude mice has made such experiments feasible, and thus observations of in vivo growth and antigenic variations of transformed human cells become possible. Previous studies have shown that irradiated human CMV retains ability to transform hamster embryo fibroblasts (l) and that the transformed cells are oncogenic in newborn hamsters. Recently, we reported that a human CMV of prostatic origin established a long-term persistent infection in HEL cells in vitro (2). After a number of cell culture passages, foci of short fibroblastoid and epithelioid cells developed, and two cell lines (designated CMV-Mj-HEL-l and CMV-Mj-HEL-2) were obtained in which foci of infection could no longer be demonstrated and from which virus could not be rescued. The cells ceased to demonstrate contact inhibition. CMV-specific antigens were demonstrated in both cell lines by the indirect IF technique. Microcytotoxicity tests established that CMV-Mj-HEL-l and CMV-MjHEL-2 cells, but not control HEL cells, share a membrane antigen(s) with hamster cells transformed by CMV. Initial studies indicated that the CMV-Mj-HEL-l cells were oncogenic in athymic nude mice and that cells cultured in vitro from the tumor had CMV-related antigens. However, karyotypic analysis could not exclude the possibility that the HEL cells became contaminated with an established human bladder tumor cell line during the period of persistent infection with CMV and that the bladder cells were subsequently transformed with CMV. The original bladder cells were oncogenic for the athymic nude mice, and thus the oncogenicity of the CMV-Mj-HEL-l cells could be an independent phenomenon and not the consequence of CMV transformation (3). The HEL origin of the second transformed cell line (CMV-Mj-HEL-2) has been confirmed by karyotype analysis. This report confirms the oncogenicity of the CMVtransformed human cells in athymic nude mice and provides evidence that passage in vivo does not lead to loss of the CMV-specific antigens in the tumor line. Cell cultures established from these tumors remained posiVOL. 58, NO.4, APRIL 1977 Downloaded from https://academic.oup.com/jnci/article-abstract/58/4/1003/908462 by University of California, Santa Barbara user on 04 March 2018
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Cells.-CMV-Mj-P cells originating from human prostate tissue that apparently had been infected in vivo with CMV were grown in Ham's medium supplemented with 20% FCS, 0.075% sodium bicarbonate, 100 IV penicillin/ ml, and 100 JLg streptomycin/ml. The cells grew in vitro to passage levels well above those routinely attained by normal cells. After a number of passages, virus was no longer rescuable, although CMV-specific antigens and nucleic acid were continually detected in the cells (2). Cells of the CMV-Mj-HEL-l and CMV-Mj-HEL-2 lines were maintained in Ham's medium with the same supplements. Animals.-Weanling athymic homozygous (nu/nu) nude mice (NIH Swiss Webster, sixth backcross generation) were obtained from Life Sciences, St. Petersburg, Florida. Inoculation of athymic nude mice with CMV-transformed HEL cells.-Cell sheets were trypsinized with a mixture of 0.1 % trypsin and 0.02% Versene and washed by centrifugation three times in Ham's medium with 0.075% sodium bicarbonate, 100 IV penicillin/ml, and 100 JLg streptomycin/ml. The cells were resuspended in the same medium and counted. Cells were inoculated sc CMV = cytomegalovirus; HEL = human embryo lung; IF = immunofluorescence; FCS = fetal calf serum; HSV-l = herpes simplex virus type 1; HSV-2 = herpes simplex virus type 2; PARA = particle aiding replication of adenovirus; SV40 = simian virus 40; ACIF = anticomplement immunofluorescence; FITC = fluorescein isothiocyanate; IUDR = 5-iodo-2'-deoxyuridine; DMSO = dimethyl sulfoxide. 1 Received June 7, 1976; accepted September 27, 1976. 2 Supported by Public Health Service (PHS) contract NOI CP53516 from the Virus Cancer Program, Division of Cancer Cause and Prevention, National Cancer Institute (NCI), and by PHS grant CA16365 from the NCI. 3 Department of Microbiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, Hershey, Pa. 17033. 4 Departments of Pathology and Microbiology, The Milton S. Hershey Medical Center. 5 We thank Dr. J. Gruber, Office of Program Resources and Logistics, Viral Oncology Program, Division of Cancer Cause and Prevention, NCI, Bethesda, Md., for supplies of nude mice; Dr. F. O'Neill, Department of Microbiology and Pathology, University of Utah Medical Center, Salt Lake City, Utah, for karyotype analysis; and Judith Blom and Julie Miskimen, Department of Microbiology, The Milton S. Hershey Medical Center, for technical assistance. ABBREVIATIONS USED:
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into the middle of the back of newborn and weanling (2to 3-wk-old) nude mice. Autopsy and histologic examination. -Tumor-bearing animals were killed with chloroform. Tissue was removed, fixed in neutral buffered formalin, cut into 5-/L paraffin sections, and stained with hematoxylin and eosin. Preparation of tumor cell cultures.-Half of the tumors were washed three times in phosphate-buffered saline, minced into 1- to 2-mm pieces, and transferred into plastic tissue culture flasks (surface area, 28 em") in Ham's medium with 20% FCS, 0.075% sodium bicarbonate, 100 IU penicillin/ml, and 100 /Lg streptomycin/mi. The fragments attached to the surface, and the cells spread and formed a confluent cell sheet within 1 week. The tumor cell cultures were normally maintained by dividing the cells from one flask into two every second day. Stained cover slip preparations. -Tumor cells grown on cover slips were fixed in methanol for 2 minutes at room temperature and were then stained with May-Griinwald-Giemsa. Immune sera.-Human antisera to CMV were obtained from hospital patients. Immune sera "W" and "T" had indirect IF antibody titers to CMV-infected HEL cells of 1:128 and 1:256, respectively. Serum Wand serum T had titers of 1:64 to HSV-1-infected HEL cells and 1:32 to HSV-2-infected HEL cells. The HSV-2-specific hamster serum had an indirect IF antibody titer to HSV-2infected HEL cells of 1:32, and sera from PARA (defective SV40)-adenovirus 7 tumor-bearing hamster reacted with homologous tumor cells at a titer of 1:8 in the indirect IF test. All immune sera used in the indirect IF and ACIF tests were adsorbed with 108 HEL cells/rnl of undiluted serum for 1 hour at 37° C and then overnight at 4° C before use. One sample of immune serum W was adsorbed with CMV-infected HEL cells (108 cells/0.5 ml of undiluted immune serum; multiplicity of infection=0.3 plaque-forming unit/cell). Serologic tests.-Standard procedures of the indirect IF test were used for the detection of CMV-specific antigens in the transformed cells (2). Goat anti-human IgG conjugated to FITC was purchased from Cappel Laboratories, Downingtown, Pennsylvania. In the ACIF test, cover slip cultures were fixed with a mixture of equal parts of methanol and acetone for 2.5 minutes, covered
with inactivated anti-CMV serum, and incubated at 37° C for 45 minutes. After repeated washings, 2-4 U of complement was added for 45 minutes at 37° C. CMV antibody-negative human serum served as the complement source. The cells were washed again with phosphate-buffered saline and covered with goat antihuman complement serum labeled with FITC for 45 minutes at 37° C. After final washings, the cells were mounted on slides in a mixture of 1 part phosphate-buffered saline and 9 parts glycerol. Virus rescue experiments.-Several different procedures were used to rescue an infectious agent from the CMV tumor cells. Cocultivation experiments were performed by seeding 2 x 106 HEL cells into 30-ml plastic flasks. The following day, 2 x 106 tumor cells were added. A confluent monolayer of the mixed culture developed within 1-2 days. In some cocultivation experiments, 100 /Lg IUDR/ml (Calbiochem, San Diego, Calif.) was added to the culture for 3 days. HEL cells were also inoculated with medium from tumor cell cultures or extracts of the cells disrupted by sonication. Tumor cells were also subjected to UV irradiation for 4 minutes at a distance of200 mm (42 ergs/sec/rum") and then seeded into 30-ml tissue culture flasks. The irradiated cells were sometimes cocultured with HEL cells. Treatment with cytochalasin B. -Cells (0.5 x 105 ) were grown on cover slips in 60-mm petri dishes containing normal medium. When the cells were attached to the cover slips, the normal medium was replaced with medium containing 1-3 /Lgcytochalasin B/ml. The cultures were incubated in the medium for 7 days and were refed twice during this period. The cells were fixed in 3 parts of methanol and 1 part glacial acetic acid; they were then stained with May-Griinwald-Giemsa and assayed for multinucleation. Between 250 and 300 consecutive cells were scored and the data recorded. The controls received DMSO, the cytochalasin B solvent, at 0.075%. RESULTS Tumors in Athymic Nude Mice Inoculated With CMV-Transformed Cells (CMV-MJ-HEL-2)
As shown in table 1, 33 of 53 athymic nude mice inoculated 2-4 weeks after birth with different numbers of CMV-Mj-HEL-2 cells developed tumors after a mean latent period of 19 days. All of the nude mice inoculated
TABLE I.-Development of tumors in athymic nude mice inoculated with CMV-transformed HEL cells
a
Group
Age at inoculation, wk
Cell line used
Dose of cells
Passage No.
1 2 3 4 5 6 7 8 9 10
3 2 3 2 3 3 4 4 2 3
CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-HEL-2 CMV-Mj-P HEL
4xl0 7 4x10 7 4x10 7 4x10 7 107 5x10· 10· 5x105 4x10 7 2x10 7
6 8 12 13 62 62 79 79 11-75 10
Number with tumor/No. inoculated 8/14 5/5 6/14 3/6 5/5 3/3 2/3 1/3 0/85 0/10
Mean latent period, days 20 19 14 17 14 18 20 13
Histology a PDT PDT PDT PDT PDT PDT PDT PDT G
PDT=Poorly differentiated tumor cells; G=granuloma.
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with 5x 106 , 107 , 2x 107 , or 4x 107 CMV-Mj-HEL-2 cells developed tumors. Only 2 of 3 nude mice inoculated with 106 transformed cells and 1 of 3 nude mice inoculated with 5 x 105 transformed cells developed tumors. No tumors developed in animals inoculated with 105 cells during their lifetime of about 8 weeks. The average size of the tumors at 3 weeks was approximately 16x 14 mm (figs. 1,2). Tumors were rather soft and occasionally attached to underlying normal tissues. In tumors removed 6-8 weeks after cell injection, central necrotic areas were common. The tumors consisted of poorly differentiated cells, possibly of epithelial origin. The cells were small and polygonal, with large nuclei and scanty, clear cytoplasm. The cells were often embedded in an abundant collagenous matrix (fig. 3). Adjacent structures, e.g., panniculus carnosus, were rarely invaded. No tumors were observed in 10 nude mice inoculated with control HEL cells, nor did tumors develop in 85 nude mice inoculated with CMV-Mj-P cells. However, 19 nude mice of this group developed granulomas after a mean latent period of 8 days. The granulomas consisted of necrotic material, presumably derived from the inoculum, which was surrounded by intensive infiltrates of neutrophils and granulation tissue composed of fibroblasts, capillary buds, and mononuclear leukocytes. Tumor Cell Cultures
The tumor cells cultured in vitro were small and epithelioid (fig. 4). Multilayered growth developed when the cell cultures were maintained without passage after development of a confluent cell sheet. The overgrowing cells were of rounded morphology and tended to detach and float in the medium. Concentration densities of approximately 106 cells/em! were reached with transformed cells, while normal HEL cells grew to 4x 105 cells/em", Karyotype and Effect of Treatment With Cytochalasin B
The parental CMV-carrier HEL cells (CMV-Mj-P) contained 46 chromosomes, had no numerical or strucTABLE
tural markers, were female, and responded with controlled nuclear division to treatment with cytochalasin B. The CMV-Mj-HEL-2-transformed and CMV-MjHEL-2,T-l tumor cells contained 47-48 chromosomes, had no numerical but some structural markers, belonged to the same sex, but developed semi-uncontrolled nuclear division after treatment with cytochalasin B (table 2). Virus Rescue Studies
No infectious virus was recovered from the tumor cells by inoculation of cell extracts onto HEL cultures or by cocultivation with HEL cells. Irradiation with UV light (4 min at a distance of200 mm, 42 ergs/sec/rnm") or pretreatment with 100 /-Lg IUDR/ml in medium for 3 days also failed to induce infectious virus. Serologic Detection of CMV-Specific Antigens
CMV-specific membrane antigens were detected in the tumor cells in different in vitro passages by indirect IF with the use of two different human convalescent sera (fig. 5, table 3). Nuclear fluorescence was observed in most cells when the ACIF technique was employed (fig. 6). The antisera did not react with normal HEL cells. CMV-immune serum was adsorbed with CMVinfected HEL cells (l08 cells/ml immune serum) for 1 hour at 37° C and then overnight at 4° C. This adsorption largely reduced the reactivity of the immune serum for the CMV tumor cell membrane. Adsorption with normal HEL cells did not affect reactivity. HSV-2-immune sera and sera from PARA(defective SV40)-adenovirus 7 tumor-bearing hamsters did not react with CMV tumor cells (table 2). DISCUSSION
We reported previously (2) that human prostate cells, which apparently were infected in vivo with CMV, initially released the virus in cell culture. During subsequent passage of the cells, release of virus could no longer be detected. However, CMV-specific membrane antigens were still demonstrable via several imrnuno-
2.-ehromosome constitution ofCMV-carrier and CMV-transformed cells Numerical markers
Cell line
Chromosome No.
CMV-Mj_Pb CMV-Mj-HEL-2 c
47-48
CMV-Mj-HEL-2,T-ld
47-48
6 G-group chromosomes
7 D-group
chromosomes
5 B-group chromosomes
Structural markers
Sex
Comments"
CND SUND
46
1 minute 3 small 1 metacentric 1 funny D 1 minute 3 small 1 metacentric 1 funny D
SUND
e CND=controlled nuclear division after treatment with cytochalasin B; SUND=semi-uncontrolled nuclear division after treatment with cytochalasin B. b CMV-Mj carrier HEL cell line, source of CMV-transformed cells. c CMV-transformed human cells. Transformed cells developed at passage 3 after inoculation with CMV. d Tumor cell culture. Tumors were induced in weanling athymic nude mice with CMV-Mj-HEL-2 cells.
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3.-Presence ofCMV-associated antigens on the surfaces of tumor cells from athymic nude mice In vitro passage
Cells CMV-Mj-HEL-2,T-1
f
CMV-Mj-HEL-2,T-2f CMV-Mj-HEL-2,T-3f HEL control HEL plus HSV-2 SV40-transformed
10 11 23 28 71 4
11 12 7 9
Specificity of antisera a Origin of cells Group 4"
Group 5" Group 7" Hamster tumor
CMVb
CMV-a c
41-49 52 41 40 40 45 50 35 45
2-5 5 ND ND ND ND ND ND ND
+
+
HSV-2 d
+
SV40e
+h (nuclear)
Indirect IF. Human convalescent serum adsorbed with HEL cells. Numbers are percent of cells with fluorescence. C Human convalescent serum adsorbed with CMV-infected HEL cells. Numbers are percent of cells with fluorescence. ND=not done. d Hamster serum adsorbed with HEL cells. e Serum from hamster bearing PARA(defective SV40)-adenovirus 7 tumor, adsorbed with HEL cells. f Tumor cell culture. Tumors were induced in weanling athymic nude mice with CMV-transformed human cells, designated CMV-Mj-HEL-2. o See table 1. h Fixed preparation. a b
logic assays, and hybridization studies with CMV -cornplementary RNA and cell DNA indicated that an average of 12 virus genome equivalents were present per cell. The CMV-Mj-P cells lost contact inhibition by passage 23 and continued to grow, reaching passage 81 some 40 weeks after their establishment in culture. Taken together, these observations suggest that the CMV-Mj-P cells were transformed by CMV. We have not, however, been able to establish a cell line beyond passage 86, and no tumors have been induced with these cells in athymic nude mice. Lang et al. (4) reported that CMV-infected human cells may acquire a morphology reminiscent of transformed cells prior to lysis. Thus the loss of contact inhibition, normally an indicator of transformation, may be misleading. However, it is also known that human transformed cells do not invariably induce tumors in nude mice (5). In subsequent experiments, HEL cells which were infected with a low multiplicity of CMV-Mj virions developed a long-term persistent infection with continuous development of CMV-specific nuclear and cytoplasmic antigens and release of infectious virus. During passage, some of these cultures went into crisis; shortly thereafter, foci of short fibroblastoid and epithelioid cells emerged and a confluent monolayer formed within 1 week. During subsequent passage of these cells, release of virus could no longer be detected. However, CMV-specific antigens continued to be synthesized. The CMV-Mj-HEL-l and CMV-Mj-HEL-2 cells lost contact inhibition and continued to grow, reaching passage 150 some 50 weeks after the first foci of transformed cells developed in the virus-carrier culture. The CMV-MjHEL-2 cells were used in these experiments because karyotypic analysis confirmed their HEL origin. Our goal was to detect direct correlation between the state of transformation by CMV and the oncogenic potential of the transformed cells in the athymic nude mice. We also
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wished to determine whether the CMV markers of the transformed cells survive the in vivo passage followed by numerous in vitro passages. In contrast to control HEL and CMV-Mj-P cells, the CMV-Mj-HEL-2 cells produced tumors when inoculated into a number of athymic nude mice. Thus the acquisition of oncogenic potential is a direct consequence of transformation by the virus. A most compelling argument against chronic infections may be made, because cells cultured in vitro from the tumor contained intracellular and membrane CMV-specific antigens detected by indirect IF as late as 71 in vitro passages after isolation. Thus passage in vivo, followed by numerous in vitro passages, did not lead to loss of the CMV-specific antigens. The tumor cells grew in vitro to high concentration densities. Previous publications have shown that cells transformed by SV40 and polyoma virus and those derived from neoplastic tissues become highly multinucleated following cytochalasin B treatment (6, 7-12). Conversely, normal and RNA virus-transformed cells become only binucleated (6, 9-12). Thus the uncontrolled nuclear division of our CMV tumor cells following cytochalasin B treatment is an indirect biologic evidence of transformation by a DNA virus. Interestingly, DNA analogue treatment was not required to obtain positive results in IF tests. The finding that CMV-immune sera reacted with the membranes of these cells and that this reactivity was specifically reduced when the immune sera were adsorbed with CMVinfected HEL cells confirms the CMV specificity of these antigens. When transplanted into athymic nude mice, human embryo fibroblasts transformed by a prostatic isolate of CMV have induced progressively growing tumors. Cell passage in vivo did not result in loss of antigens. The demonstrated capacity of CMV to transform human VOL. 58, NO.4, APRIL 1977
CMV-TRANSFORMED CELLS: TUMORIGENICITY IN ATHYMIC MICE
cells and the demonstration of CMV antigens in human prostate cancer cells (13) provide further evidence of the possible oncogenic potential of this virus in its natural host. REFERENCES (1) ALBRECHT T, RAPp F: Malignant transformation of hamster em-
(2)
(3)
(4)
(5)
bryo fibroblasts following exposure to ultraviolet-irradiated human cytomegalovirus. Virology 55:53-61, 1973 RAPp F, GEDER L, MURAsKo D, et al: Long-term persistence of cytomegalovirus genome in cultured human cells of prostatic origin. J Virol 16:982-990, 1975 GEDER L, LAUSCH R, O'NEILL F, et al: Oncogenic transformation of human embryo lung cells by human cytomegalovirus. Science 192:1134-1137, 1976 LANG DJ, MONTAGNIER L, LATARJET R: Growth in agarose of human cells infected with cytomegalovirus. J Virol 14:327-332, 1974 ROBERTS DW, WEIDANZ DP: Tumor growth in congenitally athymic (nude) mice induced by the injection of MA-160 cells. Annual Meeting of the American Society of Microbiologists, abstract E73:73, 1975
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(6) WRIGHT WE, HAYFLICK L: Formation of anucleate and multinucleate cells in normal and SV40 transformed WI-38 by cyrochalasin B. Exp Cell Res 74: 187-194, 1972 (7) O'NEILL FJ, RAPp F: Premature chromosome condensation ir hamster cells treated with cytochalasin B. Exp Cell Res 70: 226229, 1971 (8) O'NEILL FJ: Chromosome pulverization in cultured normal anc neoplastic cells treated with cytochalasin B. J Nat! Cancer Ins! 49: 1733-1738, 1972 (9) - - - : Control of nuclear division in normal but not in neoplastic mouse cells. Cancer Res 34:1070-1073,1974 (10) - - - : Limitation of nuclear division by protease inhibitors ir cytochalasin-B-treated tumor cells. J Nat! Cancer Inst 52:653657,1974 (11) KELLY F, SAMBROOK J: Differential effect of cytochalasin B or normal and transformed mouse cells. Nature [New Biol 242:217-219, 1973 (12) HIRANO A, KURIMURA T: Virally transformed cells and cytochal asin B. I. The effect of cytochalasin B on cytokinesis, karyoki nesis and DNA synthesis in cells. Exp Cell Res 89:111-120, 197~ (13) GEDER L, SANFORD EJ, ROHNER TJ, et al: Cytomegalovirus anc cancer of the prostate: In vitro transformation of human cells Cancer Ther Rep. In press
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FIGURE FIGURE FIGURE
l.-Athymic nude mouse 24 days after first sign of development of tumor following inoculation with 4x 107 CMV-Mj-HEL-2 cells. 2.-Tumor of athymic nude mouse (fig. 1) induced by CMV-Mj-HEL-2 cells. 3.-Photomicrograph of tumor induced by CMV-Mj-HEL-2 cells in athymic nude mouse. Hematoxylin and eosin. x 500
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FIGURE 4.-Photomicrograph of in vitro culture of CMV-Mj-HEL-2,T-I tumor cells of athymic nude mouse. x 500. FIGURE 5.-Photomicrograph of membrane fluorescence detected on CMV-Mj-HEL-2,T-I tumor cells (passage 15) after reaction with CMVspecific human convalescent serum and FITC-conjugated goat anti-human IgG. x 1,250. FIGURE 6.-Photomicrograph of nuclear fluorescence detected in CMV-Mj-HEL-2,T-I tumor cells (passage 22). CMV-specific human convalescent serum was used with human complement and FITC-conjugated anti-human complement goat serum in the ACIF test. x 1,250.
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