Affiliations of authors: W.-C. Liao, F. Y.-H. Wu, Division of Cancer Research, Institute of Biomedical Sciences, Academia Sinica, Taiwan, Republic of China, W.-S. W. Chang, Division of Cancer Research, Institute of Biomedical Sciences, Academia Sinica, Taiwan, and National Health Research Institutes, Taiwan, Republic of China.
Correspondence to: Felicia Y.-H. Wu, Ph.D., Division of Cancer Research, Institute of Biomedical Sciences, Academia Sinica, 128 Yen-Chiu-Yuan Rd., Section 2, Taipei 115, Taiwan, Republic of China (e-mail: bmfwu{at}ibms.sinica.edu.tw).
Aghi et al. (1) recently reported that combining cytosine deaminase (CD) and herpes simplex virus type-1 thymidine kinase (HSV-TK) prodrug-activating gene therapies resulted in synergistic anticancer effects. The 9L/CD-TK cells used were generated by transfecting parental 9L rat gliosarcoma cells with three retroviral vectors (pTLKRNL-1, pBABE-Puro, and pCD2) carrying the desired CD and HSV-TK genes. We feel it inappropriate to conclude the aforementioned synergistic effects by simply comparing the cytotoxic effects of the prodrug combinations between 9L and 9L/CD-TK cells. Some rational experiments, such as comparing the cytotoxic effects of the prodrug combinations in 9L/CD-TK and 9L cells with three control plasmids, should be included to ensure that the observed anticancer effects were indeed enhanced by the combined CD/HSV-TK treatment and not by other unexpected effects from plasmids or antibiotic stress during the process of clone selection. The authors speculated that these synergistic effects are due to enhancement of ganciclovir phosphorylation by 5-fluorouracil. To support their hypothesis, two direct approaches can be considered. First, the effect of 5-fluorouracil on the phosphorylation of ganciclovir in 9L/TK cells can be tested to clarify the cross-talk of the combined gene therapies. Second, the bystander effect can be analyzed by examining the toxicity of the prodrug combinations in control cells exposed to the conditioned medium from 9L/CD-TK cells treated with 5-fluorocytosine. Finally, the traditional combination effect analyses [the Loewe isobologram (2) and the Chou-Talalay multiple drug-effect method (3)] used by Aghi et al. may not be reliable enough to analyze quantitatively the interactions between two prodrug-activating systems. There are some genetic factors such as transgene expression that should be carefully put into consideration to design a proper cause-effect analysis for the combined gene therapies.
REFERENCES
1
Aghi M. Kramm CM, Chou TC, Breakefield XO, Chiocca EA.
Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine
deaminase gene therapies. J Natl Cancer Inst 1998;90:370-80.
2 Loewe S. The problem of synergism and antagonism of combined drugs. Arzneim-Forsch 1953;3:285-320.
3 Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55.[Medline]
Affiliations of authors: M. Aghi, E. A. Chiocca (Molecular Neuro-oncology Laboratories), X. O. Breakefield (Molecular Neurogenetics Unit), Massachusetts General Hospital, Boston; T.-C. Chou, Molecular Pharmacology and Therapeutic Program, Memorial-Sloan Kettering Cancer Center, New York, NY.
Correspondence to: E. Antonio Chiocca, M.D., Ph.D., Molecular Neuro-oncology Laboratories, Massachusetts General Hospital, East Bldg., CNY6, 13th St., Charlestown, MA 02119 (e-mail: Chiocca{at}Helix.MGH.Harvard.edu).
In their correspondence, Liao et al. provide four criticisms of our work. We do not agree with their statements for the following reasons:
1) Results described in Fig. 1 and on page 373 of the article were obtained by comparing the cytotoxicity mediated by ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) in 9L/CD-TK versus rat gliosarcoma 9L cells transfected with each plasmid (9L/CD, 9L/TK, and parental 9L cells). No functional differences in GCV sensitivity between the 9L/CD-TK and the 9L/TK clone and in 5-FC sensitivity between the 9L/CD-TK and the 9L/CD clone were observed. No functional differences in GCV insensitivity of parental 9L cells and the 9L/CD clone and in 5-FC insensitivity of parental 9L cells and the 9L/TK clone were observed. Additionally, 5-FC did not affect the proliferation of 9L/TK cells and GCV did not affect the proliferation of 9L/CD cells. Unexpected effects caused by the transfection and selection procedures are not likely, since the same selection conditions were employed for the clones (neomycin phosphotransferase resistance for 9L/CD-TK and 9L/CD cells and puromycin resistance for 9L/CD-TK and 9L/TK cells). Therefore, we disagree with the contention of Liao et al. that the use of parental 9L cells as a control for the combined prodrug therapy experiments is not appropriate.
2) In light of experiments reported in previously published literature
as well as the experiments that we describe at the end of the results
section of page 328, the suggested 5-fluorouracil (5-FU) experiment on
enhancement of GCV phosphorylation in 9L/TK and/or 9L/CDTK appears
superfluous, although it may provide additional confirmation of the
veracity of the proposed mechanism. Prichard et al. [(1) and
(28) of the article] and Harmenberg et al. [(2) and
(29) of the article) showed that inhibitors of thymidylate
synthetase (such as 5-FU) potentiated the antiviral effect of acyclovir
by decreasing intracellular pools of thymidine. Additional work by
Tattersall and Harrap [(3) and (32) of the article),
Ives et al. [(4) and (33) of the article), and Chen
et al. [(5) and (30) of the article) also shows that
this process occurs by relief of the feedback inhibition of mammalian
TK by deoxythymidine-5'-triphosphate and that thymidine is the
primary inhibitor of GCV phosphorylation. On page 328, we mention that
supplementation of thymidine completely abolished the increased
phosphorylation observed by treatment of 9L/CD-TK cells with 5-FC. We
are convinced that the latter experiment alone in conjunction with
published literature supports the proposed mechanism of synergy.
Nevertheless, we did perform the suggested experiment and, as expected,
found that 5-FU alone did enhance the phosphorylation of GCV in 9L/CDTK
cells (Fig. 1).
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4) The two analyses have been extensively employed in numerous publications, cited in the article. Furthermore, the finding of synergy by the employed analyses is supported/confirmed by the ganciclovir phosphorylation experiments of Fig. 7. We disagree that traditional combination analyses cannot be employed, because measurements were performed on a traditional pharmacologic variable (prodrug dose that produces an antiproliferative effect) in the context of a stably transfected (i.e., stably expressing) clonal cell line.
REFERENCES
1 Prichard MN, Prichard LE, Shipman C Jr. Inhibitors of thymidylate synthetase and dihydrofolate reductase potentiate the antiviral effect of acyclovir. Antiviral Res 1993;20:249-59.[Medline]
2 Harmenberg J. Intracellular pools of thymidine reduce the antiviral action of acyclovir. Intervirology 1983;20:48-51.[Medline]
3 Tattersall MH, Harrap KR. Changes in the deoxyribonucleoside triphosphate pools of mouse 5178Y lymphoma cells following exposure to methotrexate or 5-fluorouracil. Cancer Res 1973;33:3086-90.[Medline]
4
Ives DH, Morse PA, Potter VR. Feedback inhibition of thymidine
kinase by thymidine triphosphate. J Biol Chem 1963;238;1467-74.
5 Chen MS, Walker J, Prusoff WH. Kinetic studies of herpes simplex virus type 1-encoded thymidine and thymidylate kinase, a multifunctional enzyme. J Biol Chem 1979;254:10747-53.[Medline]
6 Bi WL, Parysek LM, Warnick R, Stambrook PJ. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy. Human Gene Ther 1993;4:725-31.[Medline]
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