Creation of a Model Line of Tumor Cells with Inducable Expression of Adenoviral E1A to Study Its Antiproliferative and Cytotoxic Properties In Vitro and In Vivo

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Abstract

Over the past decades, gene therapy based on the adenoviral E1A has proven its benefit against a number of tumor diseases, both in animal models and in clinical studies. It has been shown that in addition to its own antiproliferative activity, E1A also has the ability to enhance the cytotoxic effect of some anticancer drugs. The use of E1A in combination therapy can solve a number of problems in clinical oncology, among which the most pressing is the problem of drug-resistance of tumor cells. This work describes the establishment of a cell model based on human colorectal cancer cells HCT116 and cisplatin-resistant HCT116/C cells with doxycycline-inducible expression of adenoviral E1A. We have shown the concentration-dependent and time-dependent dynamics of E1A expression upon doxycycline treatment, and shown the antiproliferative effect of adenoviral E1A in the HCT116-E1A and HCT116/C-E1A cells in vitro in experiments assessing viability in MTT and clonogenic activity tests and in vivo in xenograft mouse models. Thus, as a result of our work, a model was created to explore the antiproliferative and sensitizing properties of E1A in platinum-sensitive and platinum-resistant colorectal cancer cells and to search for new approaches to anticancer therapy both in vitro and in vivo. The resulting cell line is a convenient model for selecting the most promising combinations of cytostatic drugs with E1A-based gene therapy.

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About the authors

A. V. Morshneva

Institute of Cytology, Russian Academy of Sciences

Email: marie.igotti@gmail.com
Russian Federation, 194064, St-Petersburg

A. M. Kozlova

Institute of Cytology, Russian Academy of Sciences

Email: marie.igotti@gmail.com
Russian Federation, 194064, St-Petersburg

O. O. Gnedina

Institute of Cytology, Russian Academy of Sciences

Email: marie.igotti@gmail.com
Russian Federation, 194064, St-Petersburg

M. V. Igotti

Institute of Cytology, Russian Academy of Sciences

Author for correspondence.
Email: marie.igotti@gmail.com
Russian Federation, 194064, St-Petersburg

References

  1. Baluchamy S., Sankar N., Navaraj A., Moran E., Thimmapaya B.
  2. Berhane S., Aresté C., Ablack J. N., Ryan G. B., Blackbourn D. J., Mymryk J. S., Turnell A. S., Steele J. C., Grand R. J.A.
  3. Bernhard E. J., Hagner B., Wong C., Lubenski I., Muschel R. J
  4. Chakraborty A. A., Tansey W. P
  5. Chang J. Y., Xia W., Shao R., Sorgi F., Hortobagyi G. N., Huang L., Hung M. C
  6. Chang Y.-W., Hung M.-C., Su J.-L.
  7. Cook J. L., Routes J. M
  8. Davis J. J., Wang L., Dong F., Zhang L., Guo W., Teraishi F., Xu K., Ji L., Fang B.
  9. Deng J., Kloosterbooer F., Xia W., Hung M.-C.
  10. Ferrari R., Su T., Li B., Bonora G., Oberai A., Chan Y., Sasidharan R., Berk A. J., Pellegrini M., Kurdistani S. K
  11. Frisch S. M
  12. Frisch S. M., Dolter K. E
  13. Frisch S. M., Reich R., Collier I. E., Genrich L. T., Martin G., Goldberg G. I
  14. Gerstberger S., Jiang Q., Ganesh K.
  15. Gu J., Fang B.
  16. Hendrickx R., Stichling N., Koelen J., Kuryk L., Lipiec A., Greber U. F
  17. Hofer A., Crona M., Logan D. T., Sjöberg B.-M.
  18. Hung M. C., Hortobagyi G. N., Ueno N. T.
  19. Ikeda M. A., Nevins J. R
  20. Konopka K., Spain C., Yen A., Overlid N., Gebremedhin S., Düzgüneş N.
  21. Liao Y., Zou Y.-Y., Xia W.-Y., Hung M.-C.
  22. Marthaler A. G., Adachi K., Tiemann U., Wu G., Sabour D., Velychko S., Kleiter I., Schöler H. R., Tapia N.
  23. Mendoza G., González-Pastor R., Sánchez J. M., Arce-Cerezo A., Quintanilla M., Moreno-Bueno G., Pujol A., Belmar-López C., de Martino A., Riu E., Rodriguez T. A., Martin-Duque P. 2023. The E1a adenoviral gene upregulates the Yamanaka factors to induce partial cellular reprogramming. Cells. V. 12. P. 1338.
  24. Pelka P., Ablack J. N.G., Fonseca G. J., Yousef A. F., Mymryk J. S
  25. Pelka P., Miller M., Cecchini M., Yousef A. F., Bowdish D., Dick F., Whyte P., and Mymryk J. S
  26. Pleshkan V. V., V P.V., Alekseenko I. V., V A.I., Zinovyeva M. V., V Z.M., Vinogradova T. V., V V.T., Sverdlov E. D., D S.E.
  27. Radke J. R., Cook J. L https://doi.org/10.1038/cddis.2016.445
  28. Radko S., Jung R., Olanubi O., Pelka P. https://doi.org/10. 1371/journal.pone.0140124
  29. Rao L., Debbas M., Sabbatini P., Hockenbery D., Korsmeyer S., White E.
  30. Rein D. T., Breidenbach M., Nettelbeck D. M., Kawakami Y., Siegal G. P., Huh W. K., Wang M., Hemminki A., Bauerschmitz G. J., Yamamoto M., Adachi Y., Takayama K., Dall P., Curiel D. T
  31. Saito Y., Sunamura M., Motoi F., Abe H., Egawa S., Duda D. G., Hoshida T., Fukuyama S., Hamada H., Matsuno S.
  32. Sánchez-Prieto R., Quintanilla M., Cano A., Leonart M. L., Martin P., Anaya A., Ramón y Cajal S.
  33. Shisler J., Duerksen-Hughes P., Hermiston T. M., Wold W. S., Gooding L. R
  34. Singhal G., Leo E., Setty S. K.G., Pommier Y., Thimmapaya B.
  35. Sunamura M., Yatsuoka T., Motoi F., Duda D. G., Kimura M., Abe T., Yokoyama T., Inoue H., Oonuma M., Takeda K., Matsuno S.
  36. Tiainen M., Spitkovsky D., Jansen-Dürr P., Sacchi A., Crescenzi M.
  37. Van Houdt W. J., Haviv Y. S., Lu B., Wang M., Rivera A. A., Ulasov I. V., Lamfers M. L.M., Rein D., Lesniak M. S., Siegal G. P., Dirven C. M.F., Curiel D. T., Zhu Z. B
  38. White E.
  39. Whyte P., Ruley H. E., Harlow E.
  40. Yamaguchi H., Chen C.-T., Chou C.-K., Pal A., Bornmann W., Hortobagyi G. N., Hung M.-C.
  41. Yu D., Wolf J. K., Scanlon M., Price J. E., Hung M. C
  42. Zemke N. R., Hsu E., Barshop W. D., Sha J., Wohlschlegel J. A., Berk A. J https://doi.org/10.1128/jvi.00993-23
  43. Zhu Z. B., Makhija S. K., Lu B., Wang M., Kaliberova L., Liu B., Rivera A. A., Nettelbeck D. M., Mahasreshti P. J., Leath C. A., Yamamoto M., Alvarez R. D., Curiel D.

Supplementary files

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2. 1. The effect of doxycycline (Dox) on the expression of the e1a gene under a Tet-regulated promoter. a — Western blot with antibodies against the adenovirus E1A protein from extracts of different HCT116-E1A cell clones, untreated (–) or treated (+) with 1 mcg/ml of Dox for 18 hours; HEK293 cell extract was used as a positive control for E1A-expressing cells. b, c — Western blots with antibodies against the adenovirus E1A protein from HCT116-E1A cell extracts, untreated (–) or treated with Dox in various concentrations: 0.1—1 mcg/ml for 18 hours (b) or 1 mcg/ml for various times (3-48 hours; c).

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3. 2. The effect of doxycycline (Dox) on the viability of cells with and without induced E1A expression (HCT116-E1A and HCT116/C-E1A cells). The results of the MTT test are presented in the form of graphs of changes in optical density, directly proportional to cell viability, which was evaluated relative to the control taken as 100%. The cells were left untreated (Dox–) or treated with 1 mcg/ml of Dox for 48-72 hours (Dox+). Here and in Fig. 3, 4, the average values and their standard errors from 3-5 independent experiments are presented; the values for Dox– and Dox+ were compared in pairs, the differences are significant at p < 0.05 (*, Mann test‒Whitney).

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4. 3. E1A reduces the clonogenic activity of colorectal cancer cells. a, b — Photographs of cups with cell clones colored crystal purple. HCT116 or HCT116-E1A cells were dispersed in clonal density and allowed to form colonies for 14 days in the absence of doxycycline (Dox‒) or in its presence (Dox+) at a concentration of 1 (a) or 0.5 (b) micrograms/ml in the culture medium. HCT116 cells were used as a control to analyze the possible toxic effects of doxycycline. b, d are Diagrams of the average values of clonogenic activity of HCT116 and HCT116—E1A cells relative to the values of control untreated cells (Dox-), taken as 100 %.

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5. 4. E1A slows down tumor growth in xenographic models in Balb/c-Nude mice with inoculated HCT116-E1A human colorectal cancer cells. a —Western blot of total proteins isolated from tumors, as well as from the spleen of mice treated with drinking water with doxycycline (Dox+), or not treated with it (Dox‒), with antibodies against caspase 9, α-tubulin and E1A; extracts of HCT116-E1A cells were used as positive controls, treated with 1 mcg/ml of Dox for 24 hours, HCT116 cell extracts without the E1A construct were used as a negative. b — Diagrams of tumor volume growth over 20 days, depending on the presence of Dox in drinking water. The b — Scheme of the in vivo experiment design shows the duration of the experiment and at what time (day) the volume of tumor V was measured after inoculation. d — Diagrams of tumor volume increase, expressed as a percentage of the volume of tumors of the previous measurement; light columns — tumor volume increase in control group mice that did not receive doxycycline; dark — in mice treated with doxycycline (2 mg/ml) from day 14 of the experiment.

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