Proc. Nati. Acad. Sci. USA Vol. 74, No. 6, pp. 2574-2578, June 1977
Microbiology
Enhanced oncogenic behavior of human and mouse cells after cellular hybridization with Burkitt tumor cells (Epstein-Barr virus/epithelial Burkitt hybrid cells/growth characteristics/nude mice)
RONALD GLASER*, DHARAM V. ABLASHIt, MEIHAN NONOYAMA*, WERNER HENLE§, AND JOHN EASTONt Department of Microbiology, The Milton S. Hershey Medical Center and Specialized Cancer Research Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; t Viral Oncology Program, National Cancer Institute, Bethesda, Maryland 20014; TDepartment of Micro iology, RushPresbyterian-St. Luke's Medical Center, 1753 West Congress Parkway, Chicago, Illinois 60612; and § The Joseph Stokes, Jr. Research Institute at the Children's Hospital of Philadelphia and School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
*
Contributed by Werner Henle, April 6, 1977
ABSTRACT Studies were made of the expression of the Epstein-Barr virus (EBV) in somatic hybrids of Burkitt tumor cells and human or mouse cells to determine whether EBV genetic information associated with the capacity to transform leukocytes of human and non-human primates could be maintained and expressed in nonlymphoblastoid cells. Data obtained thus far suggest that at least one characteristic associated with cellular transformation (loss of contact inhibition) is expressed only in nonlymphoblastoid cells in which the EBV genome is maintained. In addition, we have demonstrated that human epithelial/Burkitt hybrid cells (D98/HR-1 and D98/Raji) are more oncogenic in nude (athymic) mice than are cells of the human epithelial parental line, D98, or one of the Burkitt lymphoblastoid parent cell lines (Raji); the HR-1 Burkitt parent cell line was as oncogenic as the hybrid cell lines but the time required to induce tumors was much longer. Thus, human epithelial cells show alteration of growth properties in vitro and in vivo after cellular hybridization with Burkitt tumor cells.
In previous studies, cultured Burkitt tumor cells of the PsJ-HR-1 (HR-1) and Raji cell lines were fused to human D98/AH2 cells (D98), using inactivated Sendai virus (1, 2). The resulting hybrid cells, designated D98/HR-1 and D98/Raji, have been used for studies of repression and induction of Epstein-Barr virus (EBV) (3,4). We have used such cells to examine the growth properties of Burkitt hybrid cells in comparison to the nonlymphoblastoid parent cell line, and to determine whether EBV genetic information associated with the capacity to transform leukocytes in vitro can be maintained and expressed in nonlymphoblastoid cells. We had previously shown that human/Burkitt (D98/ HR-1) hybrid cells exhibit growth characteristics in culture that differ from those of the D98 parental cell line. The hybrid cells and the D98 parent were epithelial in morphology; however, the D98/HR-1 cells had lost contact inhibition, as indicated by the formation of multilayered foci. This was not true for D98 cells, which did not form foci and were contact inhibited (5). In addition, we demonstrated that the cloning efficiency of the two clones of D98/HR-1 cells tested was approximately three times greater than that of D98 cells (6). We now present data obtained from additional studies in vitro and in vivo with mouse/Burkitt and human/Burkitt hybid cells which demonstrate further that growth properties of a mouse or human cell can be altered after cell hybridization with Burkitt tumor cells.
D98/Raji CL4 cells, were maintained in hypoxanthine/aminopterin/thymidine (HAT) selective medium (1, 7) consisting of Eagle's medium supplemented with 10% fetal calf serum, streptomycin at 100 ,ug/ml, penicillin at 100 units/ml, mycostatin at 10 units/ml, Fungizone at 1 ,ug/ml, 0.75% NaHCO3, 0.1 mM hypoxanthine, 0.4 ,uM aminopterin, 16,M thymidine, and 0.106 uM glycine. Raji and HR-I cells were maintained in RPMI 1640 medium plus 10% fetal calf serum. Assay for Epstein-Barr-Associated Antigens. Mouse/ Burkitt and human/Burkitt hybrid cells were grown on glass coverslips. The cells were fixed in acetone for 3 min at room temperature. The anticomplement immunofluorescence technique was used as described (8, 9) for detection of the nuclear antigen (EBNA) and indirect immunofluorescence was used for viral capsid antigen (VCA) and early antigen (EA) (10). DNA-RNA Hybridization. The methods used to prepare EBV DNA and complementary RNA (cRNA) have been described in detail, as have the assays for EBV DNA in lymphoblastoid cells (11) and somatic cell hybrids (12). A background count of 250 cpm [DNA prepared from Simpson, an EBV-negative lymphoblast cell line (13)] was subtracted from the cpm obtained from the CLiD/Raji hybrids. The cpm of hybridized material is expressed per 50 ,ug of DNA and the number of EBV genome equivalents per cell was calculated using 50 genome equivalents in Raji cells as the standard. Fifty genome equivalents (Raji) correspond to 1592 cpm of DNAcRNA hybrids after subtraction of background, i.e., normal (Simpson) cell DNA and filters. Studies with nude Mice. Five-week-old random-mated homozygous nude (nu/nu) athymic mice were obtained from the National Cancer Institute Frederick Cancer Research Center, Litton Bionetics Animal Farm, Frederick, MD. Mice were inoculated subcutaneously with 1.0 X 106 viable D98, D98/HR-1, D98/Raji, Raji, CLiD, or CLiD/Raji cells in 0.1 ml of RPMI 1640 medium. The D98 and CLiD cells were found to be negative for EBNA and EBV DNA (9, 12). The size of the tumors was obtained by measuring the length X width X height of each tumor in each group, then obtaining an average size for a given time after inoculation. RESULTS Growth Properties of CLID/Raji Hybrid Cells. Thymidine kinase-deficient mouse CLID cells were fused with Raji cells by means of inactivated Sendai virus according to previously published procedures (1, 9). The cultures were maintained in HAT selective medium; CLiD cells were used as a control. We have never observed any revertant CLiD cells growing in HAT medium, and in these experiments all unhybridized CLiD cells
MATERIAL AND METHODS Cells. Uncloned mouse/Burkitt hybrid cells (CLLD/Raji) and human/Burkitt hybrid cells, D98/HR-1 clone 8 (CL8) and Abbreviations: EBV, Epstein-Barr virus; HAT, hypoxanthine/aminopterin/thymidine; EBNA, Epstein-Barr-associated nuclear antigen; EA, early antigen; VCA, virus capsid antigen; cRNA, complementary RNA.
2574
Microbiology: Glaser et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
2575
_U;D., _
J
vr~~~~~~;x
,.h
5z:^~~~~~~~~~~~~~~1
(a
! *
* *S :nd-,. *%.6'O*
*
g
ib~~~~~~~~j
* is s *as :§
-,
.
A.
a
FIG. 1. (A) Photomicrograph of CLiD/Raji cells, passage level 2, in which EBV markers were detected. Note multilayered foci. (X290.) (B) Photomicrograph of CLlD/Raji cells, passage level 7, in which no EBV markers were detected. Note continued difference in morphology of hybrid cells as compared to CL1D cells and the absence of multilayered foci. (X290.)
were dead within 2 weeks of exposure to HAT medium. In contrast, the CLID/Raji hybrids grew well in HAT selective medium at passage levels 2 and 7, the levels at which assays for EBV-specific markers were performed. As previously described, CLID cells were contact inhibited, as demonstrated by the fact that they grew as a monolayer without formation of multilayered foci, even when the cell sheet was 100% confluent (9). When CLID/Raji cells were examined at passage level 2 they failed to show contact inhibition and formed extensive multilayered foci, even at low cell densities (Fig. 1). However, when CLiD/Raji cells were examined at passage level 7, they still differed morphologically from CLID cells, a phenomenon consistent with cell hybridization (confirmed also by chromosome analysis), but were now contact inhibited, i.e., no multilayered foci were observed (Fig. 1). Presence of EBV-Specific Antigens in CLID/Raji Hybrid Cells. The CLiD/Raji cells that grew out in HAT selective medium were examined for EBV-specific antigens at passage level 2 by immunofluorescence techniques. They were negative for VCA and EA but EBNA was detected in 100% of the cells in some areas of the examined slides. When CLID/Raji cells were reexamined for EBNA at passage level 7, they were found to be negative (Table 1). Assay for EBV DNA in CLID/Raji Hybrid Cells. To determine whether EBV DNA was present in CLiD/Raji cells, DNA-cRNA hybridization assays were performed on cells at passage levels 2 and 7. Because DNA-DNA hybridization requires tenfold more cells than does DNA-cRNA hybridization,
and because loss of human chromosomes (and presumably the EBV genome) occurs rapidly and preferentially in mouse/ human hybrid cells (9, 14), we used the DNA-cRNA assay to overcome the time factor. It should be noted, however, that values obtained previously by DNA-cRNA hybridization were very close to yalues obtained by DNA-DNA reassociation kinetics (15). In addition, there is also a good association between the presence of the EBV genomes (detectable by nucleic acid hybridization) and the presence of EBNA (9, 16). After subtraction of 250 cpm (backgrounds were determined with Simpson cell DNA), it was found that CLID/Raji cells at passage level 2 contained 17 EBV genome equivalents per cell. By passage level 7, no EBV DNA was detectable (Table 1). Tumor Induction in nude Mice. To determine whether the change of growth properties observed in EBV genome-positive epithelial/Burkitt hybrid cells in vitro (5, 6) would be expressed also in vivo, nude mice were inoculated with 1 X 106 D98, Raji, D98/HR-1, or D98/Raji cells. All but Raji cells induced tumors in nude mice. There were, however, differences in the incidence and the time of appearance of tumors. In the first set of experiments (covering a period of 70 days) 92% (23/25) of the mice inoculated with D98/HR-1 cells developed tumors at or near the site of inoculation by 13 days after inoculation, whereas only 45% (9/20) of the animals inoculated with D98 cells developed tumors, which started to appear 22 days after inoculation (Table 2). The average size of the tumors induced by both D98 and D98/HR-1 cells varied significantly when measured 5
Table 1. Assay for EBV-specific antigens by immunofluorescence and EBV DNA by DNA-cRNA hybridization*
4-
Cell linepassage level
EA
CL1D/Raji-2 CL1D/Raji-7 Raji Simpson*
VCA EBNA +
-
-
-
-
-
-
+
cRNA
Estimated
hybridized, cpm/ 50,gg DNA
equivalents/
547 14 1592 0
genome
cell 17
Microbiology
Enhanced oncogenic behavior of human and mouse cells after cellular hybridization with Burkitt tumor cells (Epstein-Barr virus/epithelial Burkitt hybrid cells/growth characteristics/nude mice)
RONALD GLASER*, DHARAM V. ABLASHIt, MEIHAN NONOYAMA*, WERNER HENLE§, AND JOHN EASTONt Department of Microbiology, The Milton S. Hershey Medical Center and Specialized Cancer Research Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; t Viral Oncology Program, National Cancer Institute, Bethesda, Maryland 20014; TDepartment of Micro iology, RushPresbyterian-St. Luke's Medical Center, 1753 West Congress Parkway, Chicago, Illinois 60612; and § The Joseph Stokes, Jr. Research Institute at the Children's Hospital of Philadelphia and School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
*
Contributed by Werner Henle, April 6, 1977
ABSTRACT Studies were made of the expression of the Epstein-Barr virus (EBV) in somatic hybrids of Burkitt tumor cells and human or mouse cells to determine whether EBV genetic information associated with the capacity to transform leukocytes of human and non-human primates could be maintained and expressed in nonlymphoblastoid cells. Data obtained thus far suggest that at least one characteristic associated with cellular transformation (loss of contact inhibition) is expressed only in nonlymphoblastoid cells in which the EBV genome is maintained. In addition, we have demonstrated that human epithelial/Burkitt hybrid cells (D98/HR-1 and D98/Raji) are more oncogenic in nude (athymic) mice than are cells of the human epithelial parental line, D98, or one of the Burkitt lymphoblastoid parent cell lines (Raji); the HR-1 Burkitt parent cell line was as oncogenic as the hybrid cell lines but the time required to induce tumors was much longer. Thus, human epithelial cells show alteration of growth properties in vitro and in vivo after cellular hybridization with Burkitt tumor cells.
In previous studies, cultured Burkitt tumor cells of the PsJ-HR-1 (HR-1) and Raji cell lines were fused to human D98/AH2 cells (D98), using inactivated Sendai virus (1, 2). The resulting hybrid cells, designated D98/HR-1 and D98/Raji, have been used for studies of repression and induction of Epstein-Barr virus (EBV) (3,4). We have used such cells to examine the growth properties of Burkitt hybrid cells in comparison to the nonlymphoblastoid parent cell line, and to determine whether EBV genetic information associated with the capacity to transform leukocytes in vitro can be maintained and expressed in nonlymphoblastoid cells. We had previously shown that human/Burkitt (D98/ HR-1) hybrid cells exhibit growth characteristics in culture that differ from those of the D98 parental cell line. The hybrid cells and the D98 parent were epithelial in morphology; however, the D98/HR-1 cells had lost contact inhibition, as indicated by the formation of multilayered foci. This was not true for D98 cells, which did not form foci and were contact inhibited (5). In addition, we demonstrated that the cloning efficiency of the two clones of D98/HR-1 cells tested was approximately three times greater than that of D98 cells (6). We now present data obtained from additional studies in vitro and in vivo with mouse/Burkitt and human/Burkitt hybid cells which demonstrate further that growth properties of a mouse or human cell can be altered after cell hybridization with Burkitt tumor cells.
D98/Raji CL4 cells, were maintained in hypoxanthine/aminopterin/thymidine (HAT) selective medium (1, 7) consisting of Eagle's medium supplemented with 10% fetal calf serum, streptomycin at 100 ,ug/ml, penicillin at 100 units/ml, mycostatin at 10 units/ml, Fungizone at 1 ,ug/ml, 0.75% NaHCO3, 0.1 mM hypoxanthine, 0.4 ,uM aminopterin, 16,M thymidine, and 0.106 uM glycine. Raji and HR-I cells were maintained in RPMI 1640 medium plus 10% fetal calf serum. Assay for Epstein-Barr-Associated Antigens. Mouse/ Burkitt and human/Burkitt hybrid cells were grown on glass coverslips. The cells were fixed in acetone for 3 min at room temperature. The anticomplement immunofluorescence technique was used as described (8, 9) for detection of the nuclear antigen (EBNA) and indirect immunofluorescence was used for viral capsid antigen (VCA) and early antigen (EA) (10). DNA-RNA Hybridization. The methods used to prepare EBV DNA and complementary RNA (cRNA) have been described in detail, as have the assays for EBV DNA in lymphoblastoid cells (11) and somatic cell hybrids (12). A background count of 250 cpm [DNA prepared from Simpson, an EBV-negative lymphoblast cell line (13)] was subtracted from the cpm obtained from the CLiD/Raji hybrids. The cpm of hybridized material is expressed per 50 ,ug of DNA and the number of EBV genome equivalents per cell was calculated using 50 genome equivalents in Raji cells as the standard. Fifty genome equivalents (Raji) correspond to 1592 cpm of DNAcRNA hybrids after subtraction of background, i.e., normal (Simpson) cell DNA and filters. Studies with nude Mice. Five-week-old random-mated homozygous nude (nu/nu) athymic mice were obtained from the National Cancer Institute Frederick Cancer Research Center, Litton Bionetics Animal Farm, Frederick, MD. Mice were inoculated subcutaneously with 1.0 X 106 viable D98, D98/HR-1, D98/Raji, Raji, CLiD, or CLiD/Raji cells in 0.1 ml of RPMI 1640 medium. The D98 and CLiD cells were found to be negative for EBNA and EBV DNA (9, 12). The size of the tumors was obtained by measuring the length X width X height of each tumor in each group, then obtaining an average size for a given time after inoculation. RESULTS Growth Properties of CLID/Raji Hybrid Cells. Thymidine kinase-deficient mouse CLID cells were fused with Raji cells by means of inactivated Sendai virus according to previously published procedures (1, 9). The cultures were maintained in HAT selective medium; CLiD cells were used as a control. We have never observed any revertant CLiD cells growing in HAT medium, and in these experiments all unhybridized CLiD cells
MATERIAL AND METHODS Cells. Uncloned mouse/Burkitt hybrid cells (CLLD/Raji) and human/Burkitt hybrid cells, D98/HR-1 clone 8 (CL8) and Abbreviations: EBV, Epstein-Barr virus; HAT, hypoxanthine/aminopterin/thymidine; EBNA, Epstein-Barr-associated nuclear antigen; EA, early antigen; VCA, virus capsid antigen; cRNA, complementary RNA.
2574
Microbiology: Glaser et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
2575
_U;D., _
J
vr~~~~~~;x
,.h
5z:^~~~~~~~~~~~~~~1
(a
! *
* *S :nd-,. *%.6'O*
*
g
ib~~~~~~~~j
* is s *as :§
-,
.
A.
a
FIG. 1. (A) Photomicrograph of CLiD/Raji cells, passage level 2, in which EBV markers were detected. Note multilayered foci. (X290.) (B) Photomicrograph of CLlD/Raji cells, passage level 7, in which no EBV markers were detected. Note continued difference in morphology of hybrid cells as compared to CL1D cells and the absence of multilayered foci. (X290.)
were dead within 2 weeks of exposure to HAT medium. In contrast, the CLID/Raji hybrids grew well in HAT selective medium at passage levels 2 and 7, the levels at which assays for EBV-specific markers were performed. As previously described, CLID cells were contact inhibited, as demonstrated by the fact that they grew as a monolayer without formation of multilayered foci, even when the cell sheet was 100% confluent (9). When CLID/Raji cells were examined at passage level 2 they failed to show contact inhibition and formed extensive multilayered foci, even at low cell densities (Fig. 1). However, when CLiD/Raji cells were examined at passage level 7, they still differed morphologically from CLID cells, a phenomenon consistent with cell hybridization (confirmed also by chromosome analysis), but were now contact inhibited, i.e., no multilayered foci were observed (Fig. 1). Presence of EBV-Specific Antigens in CLID/Raji Hybrid Cells. The CLiD/Raji cells that grew out in HAT selective medium were examined for EBV-specific antigens at passage level 2 by immunofluorescence techniques. They were negative for VCA and EA but EBNA was detected in 100% of the cells in some areas of the examined slides. When CLID/Raji cells were reexamined for EBNA at passage level 7, they were found to be negative (Table 1). Assay for EBV DNA in CLID/Raji Hybrid Cells. To determine whether EBV DNA was present in CLiD/Raji cells, DNA-cRNA hybridization assays were performed on cells at passage levels 2 and 7. Because DNA-DNA hybridization requires tenfold more cells than does DNA-cRNA hybridization,
and because loss of human chromosomes (and presumably the EBV genome) occurs rapidly and preferentially in mouse/ human hybrid cells (9, 14), we used the DNA-cRNA assay to overcome the time factor. It should be noted, however, that values obtained previously by DNA-cRNA hybridization were very close to yalues obtained by DNA-DNA reassociation kinetics (15). In addition, there is also a good association between the presence of the EBV genomes (detectable by nucleic acid hybridization) and the presence of EBNA (9, 16). After subtraction of 250 cpm (backgrounds were determined with Simpson cell DNA), it was found that CLID/Raji cells at passage level 2 contained 17 EBV genome equivalents per cell. By passage level 7, no EBV DNA was detectable (Table 1). Tumor Induction in nude Mice. To determine whether the change of growth properties observed in EBV genome-positive epithelial/Burkitt hybrid cells in vitro (5, 6) would be expressed also in vivo, nude mice were inoculated with 1 X 106 D98, Raji, D98/HR-1, or D98/Raji cells. All but Raji cells induced tumors in nude mice. There were, however, differences in the incidence and the time of appearance of tumors. In the first set of experiments (covering a period of 70 days) 92% (23/25) of the mice inoculated with D98/HR-1 cells developed tumors at or near the site of inoculation by 13 days after inoculation, whereas only 45% (9/20) of the animals inoculated with D98 cells developed tumors, which started to appear 22 days after inoculation (Table 2). The average size of the tumors induced by both D98 and D98/HR-1 cells varied significantly when measured 5
Table 1. Assay for EBV-specific antigens by immunofluorescence and EBV DNA by DNA-cRNA hybridization*
4-
Cell linepassage level
EA
CL1D/Raji-2 CL1D/Raji-7 Raji Simpson*
VCA EBNA +
-
-
-
-
-
-
+
cRNA
Estimated
hybridized, cpm/ 50,gg DNA
equivalents/
547 14 1592 0
genome
cell 17
