REPORTS
Yoshiaki Yum,* Yasuo Kondo, (6-8). Hiroki Iga, Kouji Harada, Oral herpes simplex virus (HSV) inHitoshi Tujimoto, Tetsuo fection was reported to be an unexYanagawa, fiideo Yoshida, pected consequence of HMBA therapy Mitsunobu Sato (6,8). The frequencies of its occurrence Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of tumor cells. The effect of HMBA on cell growth and replication of herpes simplex virus (HSV) was investigated in HEp-2 epidermal cells, EVER-32 neuronal cells, K562 myeloid cells, Daudi Burkitt lymphoma cells, and CCRFCEM T-rymphoid cells. The growth of HEp-2 and IMR-32 cells was not affected by HMBA at concentrations from 0.5 through 2 mM. The growth of K562, Daudi, and CCRF-CEM cells was inhibited by HMBA at concentrations from 1 through 5 mM. When HSVinfected cells were incubated with 0.5 through 5 mM HMBA, a dose-dependent increase in virus yield was observed in HEp-2 and IMR-32 cells, but not in the other cell lines. These findings indicate that HMBA enhances the replication of HSV in epidermal and neuronal cells and that HMBA therapy may be responsible for the development of herpetic lesions. [J Natl Cancer Inst 83:186-189,1991]
Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of erythroleukemia cells (7). In vitro induction of differentiation by HMBA has been characterized in a number of murine and human leukemia and solid tumor cells (7-5). The compound is not 186
were from 20% through 31% among the cancer patients treated with HMBA, but there was no apparent relationship between the dose of HMBA and HSV infection. The etiology of this problem or possible predisposing factors are as yet undefined. It is well established that HSV persists through the lifetime of the host in a latent form in neuronal cells of ganglia and that reactivation occurs in association with a variety of stimuli, such as trauma and fever (9,70). Once reactivated, HSV migrates through nerve axons from ganglia to periphery and replicates in the epithelium, resulting in the development of a recurrent herpetic lesion (77). On the other hand, it has been shown that the process of reactivation is not necessarily followed by the development of the characteristic mucocutaneous lesions, but reactivation is frequently associated with asymptomatic virus shedding in throat, saliva, urine, cervical secretion, and tears (77). If HMBA isresponsiblefor the occurence of HSV infection, it is possible that HMBA acts directly on the induction of reactivation of the virus at the site of latent infection. Alternatively, HMBA may just promote the development of recurrent lesions by an already reactivated virus. A recent study using explant cultures of dorsal root ganglia suggested that 5 mM HMBA enhanced the reactivation of HSV from latently infected ganglia (12). However, the latter possibility that HMBA stimulates HSV repli-
cation at the peripheral site has not been investigated. The present study was conducted to examine the effect of HMBA on cell growth andreplicationof HSV in epidermal cells, neuronal cells, myeloid cells, and lymphoid cells. Evidence that HMBA enhances the productive infection of HSV in epidermal cells and neuronal cells is presented.
Materials and Methods Reagents
HMBA was purchased from Sigma Chemical Co (St Louis, Mo). Acyclovir, 9-(2-hydroxymethyl)guanine, was obtained from Nippon Wellcome KK (Osaka, Japan). The stock solutions of HMBA (100 mM) and acyclovir (20 mM) were prepared in Eaglet minimal essential medium (EMEM). Cell Lines HEp-2 human epidermal carcinoma cells, IMR-32 human neuroblastoma cells, K562 human chronic myelogenous leukemia cells, Daudi Burkitt lymphoma cells, and CCRF-CEM T-lymphoblastoid leukemia cells were obtained from Flow Laboratories, Rockville, Md. HEp-2 cells were grown in EMEM supplemented with 10% calf serum (CS) and 2 mM L-glutamine. IMR-32 cells were grown in EMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and nonessential amino acids (Flow Laboratories). K562, Daudi, and CCRF-CEM cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U of
Received July 16,1990; revised September 17,1990; accepted November 5, 1990. Supported in part by a Grant-in-Aid from the Ministry of Education, Science, and Culture ofJapan. Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, Tokushima, Japan. * Correspondence to: Yoshiaki Mira, MD, Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770, Japan.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 2, 2015
Enhanced Replication of Herpes Simplex Virus by Hexamethylene Bisacetamide
cytotoxic at concentrations that induce differentiation and is a good candidate for use as an inducer of differentiation in human cancer (4). Phase I clinical and pharmacokinetic studies revealed that plasma HMBA levels in the range of 2 mM could be achieved before thrombocytopenia, metabolic acidosis, and neurotoxicity became dose limiting
penicillin per milliliter, and 100 ug of streptomycin per milliliter. For Vero monkey kidney cells, EMEM containing 5% CS and 2 mM L-glutamine was used. Cells were maintained at 37"C in an atmosphere of 5% CO2 and 95% air. Cell viability was examined by the trypan blue dye exclusion test. Virus Infection and Virus Assay
Cell line
rMR-32
K562
Daudi
CCRF-CEM
HMBA ratiori, mW
0 0.5 1.0 2.0 5.0 0 0.5 1.0 2.0 5.0 0 0.5 1.0 2.0 5.0 0 0.5 1.0 2.0 5.0
Vol. 83, No. 3, February 6, 1991
Day3 22.8 ±3.1 23.1 ± 1.3 22.6 ± 3.3 20.6 ± 5.3 10.3 ±2.4* 12.7 ±1.6 10.4 ± 2.0 9.3 ±2.1 8.0 ±1.7* 5.3 ± 1.4f 10.2 ±1.4 10.7 ±1.1 9.8 ±1.7 10.1 ±0.5 8.1±O.5f 61.6 ±2.5 61.7 ±5.4 47.7 ± 6.6* 17.0 ±1.5* 4.1 ±0.6*
Day5 53.1 ±7.7 52.3 ±7.3 51.4 ±4.0 50.0 ± 5.4 27.5 ± 4.4f 20.0 ±1.8 20.3 ±0.8 14.1 ±2.8*
9.0±2.5t
4.8 ±1.8* 12.1 ±2.5 10.2 ±0.6 10.6 ±2.7
3.5±0.5t
1.9±0.4| 79.1 ±7.7 77.4 ±2.0 70.0 ± 8.9 63.2 ± 5.5* 4.7 ±2.3*
*P < .05. .01.
Plaque-Reduction Assay HEp-2 cell monolayers were infected with 200 PFU of HSV-1. After an adsorption period of 30 minutes, cell monolayers were washed twice with PBS and covered with medium containing 0.3% methylcellulose and various concentrations of acyclovir. After 2 to 3 days of culture, the plaques were counted. The drug concentration required to reduce the number of plaques by 50% (IDJO) was calculated from the plot of plaque number versus drug concentration. Statistical Analysis Data are means ± SD of three determinations. The statistical significance of differences between drug-treated samples and drug-free controls was analyzed with Student's / test. The difference between means was considered significant at P < .05.
Figl. Effect of HMBA on the growth of HEp-2 cells. Twenty-four hours after plating, HEp-2 cells were incubated with 0.5 mM (A), 1 mA/(A),2mA/(ni) ) or 5 mM (•) HMBA. At various intervals, the number of viable cells was determined. Control cultures (O) were maintained without HMBA. *• = P < .01. *•* =
Mean No. of cells (X 104) ± SD
the cell cultures exposed to 5 mM HMBA, but not in those treated with less than 2 mM HMBA (Fig 1). In subsequent experiments, cell lines were exposed to various HMBA concentrations for 3 or 5 days and the number of viable cells was determined. The results are shown in Table 1. When cells were exposed to 5 mM HMBA, growth inhibition was demonstrated in all of the cell lines tested. There was no inhibition at an HMBA concentration of 0.5 mM. The lowest concentration of HMBA that induced significant growth inhibition in IMR-32, K562, Daudi, and CCRF-CEM cells in either 3 or 5 days was 5 mM, 1 mM, 2 mM, and 1 mM, respectively. Effect of HMBA Concentrations on Replication of HSV-1 and HSV-2
It was supposed that, at the initial stage of HSV reactivation, only a small number of infectious viruses would be present in the ganglia and epithelia. The Results effect of HMBA on the replication of Effect of HMBA on Cell Growth HSV was tested at a low MOI. When To examine the effect of various HEp-2 cells were infected with HSV-1 at HMBA concentrations on cell growth, an MOI of 0.002 to 0.003 PFU per cell an initial experiment was performed and cultured in the presence or absence with HEp-2 cells. Twenty-four hours of 5 mM HMBA, the cytopathic effect of after plating, HEp-2 cells were exposed HSV-1, consisting of cell rounding, apto 0.5, 1, 2, or 5 mM HMBA, and the peared 18 hours after infection in the viable cells were counted dairy for 5 days. treated cells, whereas no apparent celluA significant decrease in cell number lar changes were detected at that point in was observed between day 3 and day 5 in the drug-free culture. Continued culture
Downloaded from http://jnci.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 2, 2015
HSV type 1 (HSV-1) strain F (provided by P. G. Spear, Northwestern University, Chicago, 111) and HSV type 2 (HSV-2) strain UW-268 (13) were grown in Vero cells and the aliquots were stored at —80 ° C. The virus titer was assayed by plaque-forming ability on Vero cell monolayers as described previously (14). HEp-2 cells and IMR-32 cells were infected with HSV-1 or HSV-2 at a multiplicity of infection (MOI) of 0.002 to 0.003 plaque-forming unit (PFU) per cell. After an adsorption period of 30 minutes, cell monolayers were washed twice with Dulbecco^ phosphate-buffered saline (PBS) and covered with medium containing various concentrations of HMBA. Cultures were harvested 24 hours later, and the virus titer was measured by plaque formation on Vero cell monolayers. K562 cells were infected with HSV at an MOI of 0.01 PFU per cell. Since Daudi and CCRF-CEM cells were found to be less conducive to HSV multiplication, experiments were performed at a higher MOI (1 PFU per cell).
Table 1. Effect of HMBA on cell growth
REPORTS 187
Discussion A
B
•* = P
Yoshiaki Yum,* Yasuo Kondo, (6-8). Hiroki Iga, Kouji Harada, Oral herpes simplex virus (HSV) inHitoshi Tujimoto, Tetsuo fection was reported to be an unexYanagawa, fiideo Yoshida, pected consequence of HMBA therapy Mitsunobu Sato (6,8). The frequencies of its occurrence Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of tumor cells. The effect of HMBA on cell growth and replication of herpes simplex virus (HSV) was investigated in HEp-2 epidermal cells, EVER-32 neuronal cells, K562 myeloid cells, Daudi Burkitt lymphoma cells, and CCRFCEM T-rymphoid cells. The growth of HEp-2 and IMR-32 cells was not affected by HMBA at concentrations from 0.5 through 2 mM. The growth of K562, Daudi, and CCRF-CEM cells was inhibited by HMBA at concentrations from 1 through 5 mM. When HSVinfected cells were incubated with 0.5 through 5 mM HMBA, a dose-dependent increase in virus yield was observed in HEp-2 and IMR-32 cells, but not in the other cell lines. These findings indicate that HMBA enhances the replication of HSV in epidermal and neuronal cells and that HMBA therapy may be responsible for the development of herpetic lesions. [J Natl Cancer Inst 83:186-189,1991]
Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of erythroleukemia cells (7). In vitro induction of differentiation by HMBA has been characterized in a number of murine and human leukemia and solid tumor cells (7-5). The compound is not 186
were from 20% through 31% among the cancer patients treated with HMBA, but there was no apparent relationship between the dose of HMBA and HSV infection. The etiology of this problem or possible predisposing factors are as yet undefined. It is well established that HSV persists through the lifetime of the host in a latent form in neuronal cells of ganglia and that reactivation occurs in association with a variety of stimuli, such as trauma and fever (9,70). Once reactivated, HSV migrates through nerve axons from ganglia to periphery and replicates in the epithelium, resulting in the development of a recurrent herpetic lesion (77). On the other hand, it has been shown that the process of reactivation is not necessarily followed by the development of the characteristic mucocutaneous lesions, but reactivation is frequently associated with asymptomatic virus shedding in throat, saliva, urine, cervical secretion, and tears (77). If HMBA isresponsiblefor the occurence of HSV infection, it is possible that HMBA acts directly on the induction of reactivation of the virus at the site of latent infection. Alternatively, HMBA may just promote the development of recurrent lesions by an already reactivated virus. A recent study using explant cultures of dorsal root ganglia suggested that 5 mM HMBA enhanced the reactivation of HSV from latently infected ganglia (12). However, the latter possibility that HMBA stimulates HSV repli-
cation at the peripheral site has not been investigated. The present study was conducted to examine the effect of HMBA on cell growth andreplicationof HSV in epidermal cells, neuronal cells, myeloid cells, and lymphoid cells. Evidence that HMBA enhances the productive infection of HSV in epidermal cells and neuronal cells is presented.
Materials and Methods Reagents
HMBA was purchased from Sigma Chemical Co (St Louis, Mo). Acyclovir, 9-(2-hydroxymethyl)guanine, was obtained from Nippon Wellcome KK (Osaka, Japan). The stock solutions of HMBA (100 mM) and acyclovir (20 mM) were prepared in Eaglet minimal essential medium (EMEM). Cell Lines HEp-2 human epidermal carcinoma cells, IMR-32 human neuroblastoma cells, K562 human chronic myelogenous leukemia cells, Daudi Burkitt lymphoma cells, and CCRF-CEM T-lymphoblastoid leukemia cells were obtained from Flow Laboratories, Rockville, Md. HEp-2 cells were grown in EMEM supplemented with 10% calf serum (CS) and 2 mM L-glutamine. IMR-32 cells were grown in EMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and nonessential amino acids (Flow Laboratories). K562, Daudi, and CCRF-CEM cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U of
Received July 16,1990; revised September 17,1990; accepted November 5, 1990. Supported in part by a Grant-in-Aid from the Ministry of Education, Science, and Culture ofJapan. Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, Tokushima, Japan. * Correspondence to: Yoshiaki Mira, MD, Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770, Japan.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 2, 2015
Enhanced Replication of Herpes Simplex Virus by Hexamethylene Bisacetamide
cytotoxic at concentrations that induce differentiation and is a good candidate for use as an inducer of differentiation in human cancer (4). Phase I clinical and pharmacokinetic studies revealed that plasma HMBA levels in the range of 2 mM could be achieved before thrombocytopenia, metabolic acidosis, and neurotoxicity became dose limiting
penicillin per milliliter, and 100 ug of streptomycin per milliliter. For Vero monkey kidney cells, EMEM containing 5% CS and 2 mM L-glutamine was used. Cells were maintained at 37"C in an atmosphere of 5% CO2 and 95% air. Cell viability was examined by the trypan blue dye exclusion test. Virus Infection and Virus Assay
Cell line
rMR-32
K562
Daudi
CCRF-CEM
HMBA ratiori, mW
0 0.5 1.0 2.0 5.0 0 0.5 1.0 2.0 5.0 0 0.5 1.0 2.0 5.0 0 0.5 1.0 2.0 5.0
Vol. 83, No. 3, February 6, 1991
Day3 22.8 ±3.1 23.1 ± 1.3 22.6 ± 3.3 20.6 ± 5.3 10.3 ±2.4* 12.7 ±1.6 10.4 ± 2.0 9.3 ±2.1 8.0 ±1.7* 5.3 ± 1.4f 10.2 ±1.4 10.7 ±1.1 9.8 ±1.7 10.1 ±0.5 8.1±O.5f 61.6 ±2.5 61.7 ±5.4 47.7 ± 6.6* 17.0 ±1.5* 4.1 ±0.6*
Day5 53.1 ±7.7 52.3 ±7.3 51.4 ±4.0 50.0 ± 5.4 27.5 ± 4.4f 20.0 ±1.8 20.3 ±0.8 14.1 ±2.8*
9.0±2.5t
4.8 ±1.8* 12.1 ±2.5 10.2 ±0.6 10.6 ±2.7
3.5±0.5t
1.9±0.4| 79.1 ±7.7 77.4 ±2.0 70.0 ± 8.9 63.2 ± 5.5* 4.7 ±2.3*
*P < .05. .01.
Plaque-Reduction Assay HEp-2 cell monolayers were infected with 200 PFU of HSV-1. After an adsorption period of 30 minutes, cell monolayers were washed twice with PBS and covered with medium containing 0.3% methylcellulose and various concentrations of acyclovir. After 2 to 3 days of culture, the plaques were counted. The drug concentration required to reduce the number of plaques by 50% (IDJO) was calculated from the plot of plaque number versus drug concentration. Statistical Analysis Data are means ± SD of three determinations. The statistical significance of differences between drug-treated samples and drug-free controls was analyzed with Student's / test. The difference between means was considered significant at P < .05.
Figl. Effect of HMBA on the growth of HEp-2 cells. Twenty-four hours after plating, HEp-2 cells were incubated with 0.5 mM (A), 1 mA/(A),2mA/(ni) ) or 5 mM (•) HMBA. At various intervals, the number of viable cells was determined. Control cultures (O) were maintained without HMBA. *• = P < .01. *•* =
Mean No. of cells (X 104) ± SD
the cell cultures exposed to 5 mM HMBA, but not in those treated with less than 2 mM HMBA (Fig 1). In subsequent experiments, cell lines were exposed to various HMBA concentrations for 3 or 5 days and the number of viable cells was determined. The results are shown in Table 1. When cells were exposed to 5 mM HMBA, growth inhibition was demonstrated in all of the cell lines tested. There was no inhibition at an HMBA concentration of 0.5 mM. The lowest concentration of HMBA that induced significant growth inhibition in IMR-32, K562, Daudi, and CCRF-CEM cells in either 3 or 5 days was 5 mM, 1 mM, 2 mM, and 1 mM, respectively. Effect of HMBA Concentrations on Replication of HSV-1 and HSV-2
It was supposed that, at the initial stage of HSV reactivation, only a small number of infectious viruses would be present in the ganglia and epithelia. The Results effect of HMBA on the replication of Effect of HMBA on Cell Growth HSV was tested at a low MOI. When To examine the effect of various HEp-2 cells were infected with HSV-1 at HMBA concentrations on cell growth, an MOI of 0.002 to 0.003 PFU per cell an initial experiment was performed and cultured in the presence or absence with HEp-2 cells. Twenty-four hours of 5 mM HMBA, the cytopathic effect of after plating, HEp-2 cells were exposed HSV-1, consisting of cell rounding, apto 0.5, 1, 2, or 5 mM HMBA, and the peared 18 hours after infection in the viable cells were counted dairy for 5 days. treated cells, whereas no apparent celluA significant decrease in cell number lar changes were detected at that point in was observed between day 3 and day 5 in the drug-free culture. Continued culture
Downloaded from http://jnci.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 2, 2015
HSV type 1 (HSV-1) strain F (provided by P. G. Spear, Northwestern University, Chicago, 111) and HSV type 2 (HSV-2) strain UW-268 (13) were grown in Vero cells and the aliquots were stored at —80 ° C. The virus titer was assayed by plaque-forming ability on Vero cell monolayers as described previously (14). HEp-2 cells and IMR-32 cells were infected with HSV-1 or HSV-2 at a multiplicity of infection (MOI) of 0.002 to 0.003 plaque-forming unit (PFU) per cell. After an adsorption period of 30 minutes, cell monolayers were washed twice with Dulbecco^ phosphate-buffered saline (PBS) and covered with medium containing various concentrations of HMBA. Cultures were harvested 24 hours later, and the virus titer was measured by plaque formation on Vero cell monolayers. K562 cells were infected with HSV at an MOI of 0.01 PFU per cell. Since Daudi and CCRF-CEM cells were found to be less conducive to HSV multiplication, experiments were performed at a higher MOI (1 PFU per cell).
Table 1. Effect of HMBA on cell growth
REPORTS 187
Discussion A
B
•* = P
