Cancer Letters, i (1976) 319--326 319 © Elsevier]North-Holland, Amsterdam -- Printed in The Netherlands

ONCOGENIC TRANSFORMATION OF MAMMALIAN CELLS IN VITRO BY PROFLAVINE-PHOTOINACTIVATED HERPES SIMPLEX VIRUS TYPE 2

JUI-LIEN H. LI and FRED RAPP* Department of Microbiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 (U.S.A.)

(Revised version received 1 March 1976)

SUMMARY

Herpes simplex virus type 2 (HSV-2) was photodynamical ly inactivated by exposure to normal fluorescent light following direct t reatment with proflavine. Exposure of hamster embryo cells to appropriately inactivated virus yielded clones of non-contact-inhibited cells. The HSV-2-transformed cells were oncogenic when inoculated into newborn Syrian hamsters; resulting tumors were undifferentiated fibrosarcomas. HSV-2-specific antigens were found in the cytoplasm and on the surface of the transformed cells; however, no virus was recovered. Results further confirmed the onco- genic potential of HSV after photodynamic inactivation.

INTRODUCTION

It has been known for several years that animal viruses, such as herpes simplex viruses (HSV) and simian papovavirus 40 (SV40), will form com- plexes with certain dyes, such as neutral red, proflavine and toluidine blue, and that these complexes are subject to rapid biological inactivation when exposed to visible light in the presence of molecular oxygen [10] . Recently, this dye-light photoinactivation procedure, using neutral red or proflavine, has been applied as a therapeutic technique for the t reatment of HSV infec- tions in man [1 ,2] . It has been suggested, however, that dye-light t reatment for HSV infection in man may be clinically hazardous. Neutral red-photo- dynamically inactivated HSV and SV40 have been shown to transform hamster embryo cells in vitro [6] , and these transformed cells are oncogenic when inoculated into the original host [4] . Evidence of toluidine blue photo t rea tment of SV40 has also been reported to malignantly transform hamster kidney cells [9] . Because photoreactive proflavine is used in clinical

*Address correspondence to Dr. Fred Rapp, at the above address.

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trials, it is important to know whether this dye-light treatment of HSV is also potentially dangerous. This report examines the malignant transforming potential of HSV-2 following proflavine-photoinactivation.

M A T E R I A L S A N D M E T H O D S

For photodynamic inactivation of HSV-2, the direct treatment of virus with proflavine was used. The applied methods have been described previ- ously [ 6,7,11 ]. Briefy, proflavine was dissolved in triple distilled water to make a 1 mg/ml solution and filtered through a miUipore filter with 0.22 ~m pores. Dowex cation exchange resin, 50W-X4 (H+), 50--100 mesh form, was repeatedly washed with 0.85% NaC1 until a pH of 6.0--6.5 was reached. The solution was then packed into 13 X 100 mm tubes to a height of 35 mm, sterilized by boiling for 20 min, and washed repeatedly with sterile sodium tetraborate solution at pH 9.0 until the supernatant was constant at tha )H. The fluid was then removed from the tubes and the packed resin was ready to receive the virus-dye mixtures.

Stock viruses of HSV-2 strain 333 were diluted ten-fold with Tris buffer (pH 7.4) to 107 PFU/ml and were mixed with proflavine to a final concen- tration of 25 pg/ml. The virus-dye mixtures were then incubated at room temperature for 1 h. All procedures described were carried out in a darkroom with a safety red light. Following exposure to fluorescent light for varying periods of time (1--12 min), virus-dye samples were mixed with Dowex resin

I f I I I l I I I I

OI

- I

~-2

8

-ot - 7 I I i I I I I I I I

0 2 4 6 8 10 EXPOSURE TIME IN MINUTES

. . . . . . . . . . ~ . . . . ~_ ~ - - - ~ - - - - # . . . . ~-__~

Fig. 1. P h o t o d y n a m i c inac t iva t ion of HSV-1 ( - -o- - ) and HSV-2 (---o--) by d i rec t ly t r ea t ing virus wi th 25 /~g /ml p rof lav ine ( - - ) or wi th no dye ( . . . . ).

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to remove excess dye. The virus samples were chilled and assayed for surviving infectivity. A six log decrease in infectivity was observed 10--11 min after light t reatment of proflavine-HSV-2 samples (Fig. 1). This did not occur with dye-free control virus samples exposed to light for simiIar periods.

RESULTS AND DISCUSSION

For t rans format ion experiments, hamster embryo cells (5--6 × l 0 s ceUs/ml) were treated with proflavine-photoinactivated HSV-2 (exposed to light for 8, 10 or 12 min) at a multiplicity of 1--2 (prior to inactivation) in small test tubes and were gently shaken for 1 h at 37°C. Controls were virus-free proflavine-treated hamster cells or normal hamster embryo cells. Following adsorption, the virus-cell suspensions were plated in 60 mm plastic petri dishes a n d maintained in Dulbecco's medium with 10% fetal calf serum, 0.225% NaHCO3, and antibiotics against bacteria and fungi. Within one week, cell cultures infected with HSV-2 exposed to light for 8 min exhibited typical HSV cytophathic effect (CPE). Two weeks after the initiation of the experi- ment, these cultures were destroyed. Cellcultures treated with HSV-2 in- activated with light for 10 min were only partially destroyed. Virus exposed for 12 min did not produce any CPE in hamster cells and, by 21 days, loci of morphologically altered cells appeared. Clones picked from cell cultures treated with HSV-2 that had been inactivated with light for 10 rain did not survive more than five passages. Of 45 clones picked from cultures initiated with virus inactivated for 12 min, three clones became established cell lines.

TABLE 1

DETECTION OF VIRUS-SPECIFIC INTERNAL ANTIGENS IN PROFLAVINE-LIGHT INACTIVATED HSV-2-TRANSFORMED HAMSTER CELLS BY INDIRECT IMMUNO- FLUORESCENCE TESTS

Cell type Specificity of antisera

HSV-1 a HSV-2 b HSV-2 TB c SV40 (T antigen) d

pf-2-333-8 , -+ +- + -- pf-2-333-12 +_ _+ + -- pf-2-333-23 +- + + -- S V 4 0 - 6 0 - 1 2 e - - - - - - +

N o r m a l H E F . . . .

H E F + H S V - 1 + + + - -

HEF + HSV-2 + + + --

apooled sera from hamsters immunized with HSV-1. bpooled ser~ from hamsters immunized with HSV-2. cSera from tumor-bearing (TB)hamsters produced by cells transformed by ultraviolet- irradiated HSV-1 or neutral red-photoinactivated HSV-2. dpooled sera from hamsters bearing tumors produced by SV40-transformed cells. eHamster embryo cells transformed by neutral red-photoinactivated SV40.

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These three lines were designated pf-2-333-8, pf-2-333-12, and pf-2-333-23. Foci were also seen in the proflavine-treated hamster cell control cultures. Five clones were picked and passed into 35 mm petri dishes. The clones grew slowly after transfer and piling-up no longer occurred as in the clones picked from virus-infected cultures. These cultures did not survive 10 passages. Of the numerous clones (approximately 100) picked from control cultures in this and other studies, none has survived the 12th passage.

Newborn Syrian hamsters were inoculated subcutaneously with proflavine- treated HSV-2-transformed cells (pf-2-333-8). Palpable undifferentiated fibrosarcomas were produced 6--8 weeks after infection. The tumors were trypsinized and grown in cell culture for further characterization.

The tumor cell lines were morphologically similar to those of the original transformed cells; they consisted of irregularly shaped fibroblastoid cells

Fig. 2. Immunofluorescence photomicrograph of cytoplasmic antigens present in hamster embryo cells transformed by proflavine-photoinactivated HSV-2.

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with some giant and few epithelioid cells. The morphology did not change with passage.

No infectious HSV-2 virus has yet been isolated from these three lines of HSV-2-transformed cells. The transformed cells were, however, susceptible to superinfection with homologous virus.

The HS~-specific antigens present in the proflavine-HSV-2-transformed cells were detected by indirect immunofluorescence (IF) techniques. The cells grown on coverslips were fixed with acetone for 10 min and were reacted with HSV-specific hamster serum and fluorescein-conjugated rabbit anti-hamster gamma globulin (Table 1). The hamster sera were'obtained by immunizing with HSV-1 or HSV-2 that had been purified by sucrose gradient centrifugation. The viruses had been grown in rabbit kidney cells. Some of the sera were obtained from hamsters bearing tumors induced by cells

Fig. 3. Immunofluorescence photomicrograph of surface antigens present on hamster embryo ceUs transformed by proflavine-inactivated HSV-2.

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transformed by HSV-1 or HSV-2. The specificity of this IF reaction was investigated by comparing the antigenicity of proflavine-HSV-2-transformed cells with normal and SV40-transformed hamster cells. The transformed cells showed weak, diffuse cytoplasmic staining when reacted with antiserum to HSV-1 or HSV-2. The bright fluorescence was seen in 10--15% of the cells when reacted with HSV-specific sera from animals bearing tumors (Fig. 2). No fluorescence was found in normal hamster embryo cells or in SV40-trans- formed cells when reacted with the same sera. The transformed cells were tested for the presence of SV40 tumor antigen and were found to be negative. The indirect IF method was also used for detection of HSV-specific surface antigens present on the transformed cells. Approximately 5"10% of the cells were found to have surface fluorescence when reacted with HSV-specific tumor-bearing serum (Fig. 3). No surface fluorescence was found in control normal hamster cells or in SV40-transformed cells. The antigenic properties of tumor cells derived from the original transformed cells are currently being investigated . . . .

These results demonstrate the oncogenic potential of HSV-2 following proflavine and light photoinactivation. The results are not surprising since proflavine is a mutagen in bacteria [10] and in animal virus systems [3], and can also be a carcinogen. However, it is possible that proflavine alone will convert normal cells into malignant cells. These results further demonstrate that photodynamic inactivation of HSV with dye can differentially inactive virus lyric function and unmask the oncogenic potential of tumor viruses. Coupled with clinical studies failing to show efficacy of dye-light treatment [5,8], these results strongly mitigate against use of this form of therapy in man.

ACKNOWLEDGEMENTS

This study was conducted under Contract N01 CP 53516 within the Virus Cancer Program of the National Cancer Institute, NIH, PHS.

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