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Effect of Polar-planar compounds on the differentiation of normal human myeloid immature marrow cells
Cancer Letters, 46 (1989) 37-41 f&&or Scientific Publishers Ireland Ltd.
37
Effect of Polar-planar compounds on the differentiation of normal human myeloid immature marrow cells H.T. Hassan and J.K.H. Rees Department (U.K.)
of Haematological
Medicine,
Uniuersity
of Cambridge
Clinical School, MRC Building, Hflls Road, Cambridge
(Received 22 December 1988) (Revision received 2 March 1989) (Accepted 6 March 1989)
Summary
Polar-planar compounds which are potent differentiating agents, have been reported lo enhance the response of friend erythroleukaemic cells and other malignant cell lines to mitomycin C, cis-platinum and radiation.
of
With the upcoming phase I1 clinical trials of these polar compounds alone and in combination with cytotoxic agents, the effect of 3 polar
compounds: dimethylsulphoxide, dimethylformamide and hexamethylene bisacetamide on the differentiation of normal human myeloid immature marrow cells was studied. The results showed that a long-term exposure to these polar compounds may lead to a premature senescence of these marrow progenitors.
Keywords:
cell differentiation; dimethylformamide; dimethylsulphoxide; hexamethylene bisacetamide; myelopoiesis. Introduction
Polar-planar compounds are chemical solvents with low molecular weight, a polar hydrophilic moiety and a planar hydrophobic
Correspondence to: H.T. Hassan, Cambridge CB3 9EU, U.K.
Street,
Darwin
College,
Silver
portion [ 171. They have been reported to induce differentiation of several leukaemic and other malignant cell lines [10,23]. The induction of differentiation was associated with changes in gene expression, enzyme activities, chromatin structure, surface membrane composition and fluidity and clonogenicity of the malignant cells [ZO] and was not affected by the inhibitory factors in leukaemic serum [ 111. The exact mechanism of action of these polar compounds as differentiating agents is still unknown [ 121. Two levels of tumour cell heterogeneity are expressed: one exists at any time among subpopulations of tumour cells and the other develops with time by changing the behavioural patterns of the tumour, a phenomena known as progression [2]. In addressing approaches that might lessen this problem, it has been suggested that the induction of a more differentiated state in malignant cells by differentiating agents may minimize diversity [5]. The rational for this suggestion is that such treatment with differentiating agents can decrease the variability of response among the subpopulations of a heterogeneous tumour [9]. Recently, in vitro studies showed that such differentiation treatment when combined with cytotoxic therapy decrease the extent of variability of response and enhance the sensitivity of tumour cells towards cytotoxic drugs. Polar compounds have been reported to
0304-3835/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland I_td Published and Printed in Ireland
38
increase the sensitivity of cultured human colon tumour cells to mitomycin C and cisplatinum [4]. Dimethylformamide (DMF) has been reported to increase the sensitivity of two clones of a heterogeneous human colon adenocarcinoma cell line as well as two mouse mammary adenocarcinoma sublines to radiation and to reduce the ability of their cells to repair sublethal radiation damage [ 1,181. Also, (DMSO) has been dimethylsulphoxide reported to increase the response of cultured friend erythroleukaemic cells to radiation [7]. These results have important clinical implications. With the upcoming of phase II clinical trials of polar compounds as differentiating agents alone and in combination with conventional cytotoxic agents [3,6,8,19,22] as well as in purging for autologous bone marrow transplantation in leukaemic patients [13], it is of interest to investigate their effect on the differentiation of normal human immature marrow cells. Weimann et al. have reported that DMF induced premature senescence of murine bone marrow cell cultures [25]. In the present study, the effect of three polar compounds: dimethylsulphoxide, dimethylformamide and hexamethylene bisacetamide on the differentiation of normal human myeloid immature marrow cells was examined. The results showed that a long term exposure to these three polar compounds may lead to a premature senescence of these marrow progenitor cells. Materials and methods
Preparation of normal marrow cells Normal marrow aspirates were collected from 22 normal healthy donors, whose haematological profiles and differential count of their marrow aspirates were within the normal haematological ranges [26]. Mononuclear marrow cells were obtained by layering over Ficoll-hypaque (density, 1.077) and centrifuging for 25 min at 600 x g. Non-adherent mononuclear marrow cells were obtained from two cycles of incubation in plastic tissue culture plates, each for 1 hour at 37OC in a humidified
incubator under 5% CO,. The viability of cells as determined by trypan blue exclusion test was> 96%. Cell culture Non-adherent mononuclear marrow cells were seeded in 12-well tissue culture plates containing RPM1 1640 medium with 20% heat-inactivated (56OC for 30 min) fetal calf serum and 10% 5637-conditioned medium which was prepared as described before [21], at a concentration of 5 x lo5 cells/ml with 160 mM DMSO, 100 mM DMF or 2 mM hexamethylene bisacetamide (higher concentrations of the 3 polar compounds were cytotoxic and might kill immature cells selectively) and without as a control and incubated for 6 days at 37 OC in a humidified incubator under 5% co*. Assessment of differentiation at day 6 Morphologically, using Hyel-Romanowsky stain, the cytocentrifuge smears were prepared on day 6 of culture and at least 200 cells were scored for morphological maturation. Nitroblue tetrazolium due reduction test was carried out as described before [14] and at least 200 cells were scored for the presence of intracellular blue-black formazan granules. Results
The incubation of normal non-adherent mononuclear marrow cells with the 3 polar compounds for the entire 6 days of culture did not affect their survival (Table 1). A genuine differentiation had taken place with an absolute and not only proportional increase in both morphologically mature cells and nitroblue tetrazolium dye reducing cells (Table 1). The increase represents a true stimulation of differentiation rather than a selective killing of the immature cells since there was an increase in the total number of viable cells at day 6 compared to those cultured at day 0 (Table 1). The addition of 160 mM DMSO to cultures caused an increase of 13.5-15.2% in the number of mature granulocytes and a decrease in
l l
216 112 132 159
f f f f
10.4 18.0 18.1 14.0
Blasts, promyelocytes and myelocytes
Morphology’ l
395 318 302 336
f f f f
14.6 16.7 12.2 18.2
Metamyelocytes
(Absolute no. x 105)
229 260 244 264
f f f +
10.6 14.9 14.9 18.4
Mature Granulocytes
17 17 17 18
f f f k
1.9 1.7 1.8 1.6
Monocytes
on the differentiation of human normal myeloid immature marrow cells.
DMSO: dimethylsulphoxide, DMF: dimethylformamide, HMBA: hexamethylene bisacetamide. ‘In control and treated cultures after six days of incubation. The initial cell number on day 0 was 5 x 105. l ‘Differential count of 500 cells counted on cytocentrifuge smears of control and treated cultures on day 6. ***Mean f S.D. (n = 22).
1.8’ 1.4 1.8 1.6
8.5 7.0 6.8 7.5
Control 160 mM DMSO 100 mM DMF 2 mM HMBA
f f + f
Number of viable cells (x 105)*
Effect of Polar-planar compounds
Polar-planar compound
Table 1.
627 774 727 786
zt f f f
10.6 17.8 14.5 16.4
Nitroblue tetrazolium dye reducing cells (Absolute no. x 105)
40
immature cells of 39.5-52% compared with control cultures. The number of monocytes was not changed (Table 1). After incubation with 160 mM DMSO for 6 days, the nitroblue tetrazolium due reducing cells increased by 23.5-29.796 compared with control cultures. The addition of 100 mM DMF to cultures caused an increase of 6.7-12.646 in the number of mature granulocytes and a decrease in immature cells of 36.3-41.2% compared with control cultures. The number of monocytes was not changed (Table 1). After incubation with 100 mM DMF for 6 days, the nitroblue tetrazolium dye reducing cells increased by 13.5-15.9% compared with control cultures. The addition of 2 mM hexamethylene bisacetamide (HMBA) to cultures caused an increase of 12.6-15.1% in the number of mature granulocytes and a decrease in immature cells of 21.1-26.3% compared with control cultures. The number of monocytes was not changed (Table 1). After incubation for 6 days with 2 mM HMBA, the nitroblue tetrazolium dye reducing cells increased by 22.1-25.4% compared with control cultures. Most of the metamyelocytes and all mature granulocytes reacted positively with nitroblue tetrazolium dye. The exposure of normal human immature marrow cells to 160 mM DMSO for 6 days in culture reduced the immature cell population to 52 46 of control cultures. DMF (100 mM) and HMBA (2 mM) caused less decline of the immature cell population to 61.2% and 73.7%, respectively, of control cultures. The decline was associated with an increase of mature granulocytes after 6 days of culture with 160 mM DMSO, 100 mM DMF and 2 mM HMBA to 113.5%, 106.7% and 115 % of control cultures, respectively. Discussion
Effective control of neoplastic growth could depend upon the ability to manipulate tumour heterogeneity. Methods of directing and controlling the differentiation of tumour cells could potentially limit heterogeneity through the
conversion of tumour subpopulations into a single more differentiated phenotype [9]. Such single phenotype could be an easier target for conventional cytotoxic therapy [5]. Exposure of malignant cells to polar compounds followed by treatment with cytotoxic agents has been reported to enhance their antitumour effectiveness [ 1,4,7,18]. This simplifies therapy as less dosage and/or duration of cytotoxic drugs would be required to treat such homogenized tumour which can respond in a more uniform fashion. The membrane changes caused by polar compounds may facilitate the transport of cytotoxic drugs into the malignant cells. Also, the alterations in enzyme activities which are important in drug metabolism could make the primed tumour more responsive to the appropriate cytotoxic drug [24]. In the present study, the results showed that the 3 polar compounds: DMSO, DMF and HMBA are able to increase the number of normal human myeloid mature marrow cells and decrease the immature myeloid marrow cells after 6 days of culture. While a decrease of immature marrow cells to 52% of control in DMSO-treated cultures was associated with only 113.5% increase of mature cells, in HMBA-treated cultures a less decrease of immature marrow cells to only 73.7% of control was associated with 115% increase of mature cells. Apart from the hepatic, ocular and nervous toxicities of polar compounds, these results may indicate that long-term exposure to them could lead to a decrease in the population of normal human myeloid immature marrow cells in the order DMSO > DMF > HMBA. Although, phase I clinical trials of polar compounds have not yet shown any myelosuppression [3,6,8,19,22], the results of the present study together with those reported by Wiemann et al. [25] anticipate a premature senescence of normal immature marrow cells following long-term exposure to these agents. A novel approach to control malignant diseases would be achieved by the development of less toxic and more effective analogues of polar compounds for clinical trials [ 15,161.
41
References 1
2
3
4
5
6
7
8
9
10
11
12
Arundel, CM., Glkksman. A.S. and Leith, J.T. (1985) In vitro effects of N-N-dimethylformamide on sublethal and potenttally lethal damage recovery processes after X-radiation in heterogeneous human colon tumour cells. Cancer Res., 45,5557-5562. Calabresi, P., Dexter, D.L. and Heppner, G.H. (1979) Clinical and pharmacologic implications of cancer cell differentiation and heterogeneity. B&hem. Pharmacol., 28, 1933- 1939. Chun, H.G., Leyland-Jones, B., Hoth, D., Shoemaker, D., Wolpert-DeFillippes, M., Grieshaber, C., Cradock, J., Davidgnon, P., Moon, R., Rifktnd, R. and Wittes, R.E. (1986) Hexamethylene bisacetamide: A polar-planar compound entering clinical trials as a differentiating agent. Cancer Treat. Rep., 70,991-996. Dexter, D.L., DeFusco, D.L. and McCarthy, K. (1983) Polar solvents increase the sensitivity of cultured human colon cancer cells to &-platinum and mitomycin C. Proc. Am. Assoc. Cancer Res., 24,267. Dexter, D.L., Leith, J.T. and Crabtree, G.W. (1982) N-Ndimethylformamide induced modulation of responses of tumour cells to conventional anticancer treatment modalities. In: Maturational factors and Cancer, pp. 105112. Editor: M.A.S. Moore. Raven Press, New York. Egorin, M. J., Sigman, L.M., VanEcho, D.A., Forrest, A., Whitacre, M.Y. and Aiiner, J. (1987) Phase I clinical and pharmacological study of hexamethylene biiacetamide (NSC 95580) administrated as a five day continuous infusion. Cancer Res., 47,617-623. Einspenner, M., Boulton, J.E. and Borsa, J. (1984) Variation of radiation sensitivity of friend erythroleukaemia cells cultured in the presence of the differentiation inducer: DMSO. Rad. Res., 97.55-63. Ettinger, D.S., Grr, D.W., Rice, A.P. and Donehower, R.C. (1985) Phase I study of N-Methylformamide in patients with advanced cancer. Cancer Treat. Rep., 69, 489-593. Francis, G.E. and Pinsky, CM. (1987) Growth and differentiation control. In: Cancer chemotherapy and Biologtcal response modifiers, pp. 502-538. Editors: H.M. Pinedo, D.L. Longo and B.A. Chabner. Elsevter, Amsterdam. Hassan, H.T. (1988) Differentiation inducers of human myeloid leukaemia cell lines. Folia Haematol., 115, 887896. Hassan, H.T. and Rees, J.K.H. (1988) Effect of AML serum on the differentiation of normal myeloid progenitors and the hexamethylene b&a&amide-induced granulocyttc differentiation of the human HL-60 promyelocytic leukaemia cell line. Blood, 72,204a, 725. Hassan, H.T. (1988) Dtfferentiation induction therapy of
13
14
15
16
17
18
19
20 21
22
23
24
25
26
acute myelogenous leukaemias. Haematologia, 21, 141150. Hassan, H.T. and Rees, J.K.H. (1988) Autologous bone marrow transplantation in leukaemias and lymphomas. J Med. Res. Inst., 9,51-83. Hassan, H.T. and Rees, J.K.H. (1988) Retinoic acid alone and in combinatton with cytostne arabinoside induces differentiation of human myelomonocytic and monoblastic leukaemia cells. Haematol. Gncol., 6,39-45. Hassan, H.T. (1988) Differentiation induction therapy: An alternative for the treatment of elderly patients with acute myelogenous leukaemia. J. Clin. Exp. Gerontol., 10, 63 -73. Hassan, H.T. and Rees, J.K.H. (1989) Induction of differentiation of AML blasts in primary culture by retinoic acid + cytosine arabinoside + hexamethylene bisacetamide. Cltn. Res., 37,16A. Langdon, S.P. and Hickman, J.A. (1987) Correlation between the molecular weight and potency of polar compounds which induce the differentiation of HL-60 human promyelocytk leukaemk cells. Cancer Res., 47. 140144. Letth, J.T., Brenner, H.J. and DeWyngaert, J.K. (1981) Selective modtfication of the X-ray survival response of two mouse mammary adenocarcinoma sublines by N-N-dimethylformamide. Int. J. Rad. Gncol. Biol. Phys., 7, 943947. McVie, J.G., Ten Bokkel Huinink, W.W. and Simonetti, G. (1984) Phase I trial of N-methylformamide. Cancer Treat. Rep., 68,607-610. Marks, P.A. and Rtfkind, R.A. (1984) Differentiation modifiers. Cancer, 54.2766-2769. Myers, C.D., Katz, F.E., Joshi, G. and Millar, J.L. (1984) A ceU line secreting stimulating factors for CFU-GEMM culture. Blood, 64,152-155. Rowtnsky, E.K., Ettinger, D.S., Growchow, L.B., Brundrett, R.B., Cates, A.E. and Donehower, R.C. (1986) Phase I and pharmacological study of hexamethylene bisa&amide in patients with advanced cancer. J. Clin. Oncol., 4, 1835-1844. Sartorellt, A.C. (1985) Malignant cell dtfferentiatton as a potential therapeutic approach. Br. J. Cancer, 52, 293302. Spremulli, E.N. and Dexter, D.L. (1984) Polar solvents: A novel class of antineoplastic agents. J . Cltn. Oncol., 2,227 -241. Wiemann, M.C., Haronian, H. and Creswick, B.C. Jr. (1983) Dimethylformamide (DMF) induced premature senescence of murine bone marrow cell cultures. Proc. Am. Assoc. Cancer Res., 24,56. Wintrobe, M.M. (1981) Clinical Haematology, Lea and
Febiger, Philadelphia.
37
Effect of Polar-planar compounds on the differentiation of normal human myeloid immature marrow cells H.T. Hassan and J.K.H. Rees Department (U.K.)
of Haematological
Medicine,
Uniuersity
of Cambridge
Clinical School, MRC Building, Hflls Road, Cambridge
(Received 22 December 1988) (Revision received 2 March 1989) (Accepted 6 March 1989)
Summary
Polar-planar compounds which are potent differentiating agents, have been reported lo enhance the response of friend erythroleukaemic cells and other malignant cell lines to mitomycin C, cis-platinum and radiation.
of
With the upcoming phase I1 clinical trials of these polar compounds alone and in combination with cytotoxic agents, the effect of 3 polar
compounds: dimethylsulphoxide, dimethylformamide and hexamethylene bisacetamide on the differentiation of normal human myeloid immature marrow cells was studied. The results showed that a long-term exposure to these polar compounds may lead to a premature senescence of these marrow progenitors.
Keywords:
cell differentiation; dimethylformamide; dimethylsulphoxide; hexamethylene bisacetamide; myelopoiesis. Introduction
Polar-planar compounds are chemical solvents with low molecular weight, a polar hydrophilic moiety and a planar hydrophobic
Correspondence to: H.T. Hassan, Cambridge CB3 9EU, U.K.
Street,
Darwin
College,
Silver
portion [ 171. They have been reported to induce differentiation of several leukaemic and other malignant cell lines [10,23]. The induction of differentiation was associated with changes in gene expression, enzyme activities, chromatin structure, surface membrane composition and fluidity and clonogenicity of the malignant cells [ZO] and was not affected by the inhibitory factors in leukaemic serum [ 111. The exact mechanism of action of these polar compounds as differentiating agents is still unknown [ 121. Two levels of tumour cell heterogeneity are expressed: one exists at any time among subpopulations of tumour cells and the other develops with time by changing the behavioural patterns of the tumour, a phenomena known as progression [2]. In addressing approaches that might lessen this problem, it has been suggested that the induction of a more differentiated state in malignant cells by differentiating agents may minimize diversity [5]. The rational for this suggestion is that such treatment with differentiating agents can decrease the variability of response among the subpopulations of a heterogeneous tumour [9]. Recently, in vitro studies showed that such differentiation treatment when combined with cytotoxic therapy decrease the extent of variability of response and enhance the sensitivity of tumour cells towards cytotoxic drugs. Polar compounds have been reported to
0304-3835/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland I_td Published and Printed in Ireland
38
increase the sensitivity of cultured human colon tumour cells to mitomycin C and cisplatinum [4]. Dimethylformamide (DMF) has been reported to increase the sensitivity of two clones of a heterogeneous human colon adenocarcinoma cell line as well as two mouse mammary adenocarcinoma sublines to radiation and to reduce the ability of their cells to repair sublethal radiation damage [ 1,181. Also, (DMSO) has been dimethylsulphoxide reported to increase the response of cultured friend erythroleukaemic cells to radiation [7]. These results have important clinical implications. With the upcoming of phase II clinical trials of polar compounds as differentiating agents alone and in combination with conventional cytotoxic agents [3,6,8,19,22] as well as in purging for autologous bone marrow transplantation in leukaemic patients [13], it is of interest to investigate their effect on the differentiation of normal human immature marrow cells. Weimann et al. have reported that DMF induced premature senescence of murine bone marrow cell cultures [25]. In the present study, the effect of three polar compounds: dimethylsulphoxide, dimethylformamide and hexamethylene bisacetamide on the differentiation of normal human myeloid immature marrow cells was examined. The results showed that a long term exposure to these three polar compounds may lead to a premature senescence of these marrow progenitor cells. Materials and methods
Preparation of normal marrow cells Normal marrow aspirates were collected from 22 normal healthy donors, whose haematological profiles and differential count of their marrow aspirates were within the normal haematological ranges [26]. Mononuclear marrow cells were obtained by layering over Ficoll-hypaque (density, 1.077) and centrifuging for 25 min at 600 x g. Non-adherent mononuclear marrow cells were obtained from two cycles of incubation in plastic tissue culture plates, each for 1 hour at 37OC in a humidified
incubator under 5% CO,. The viability of cells as determined by trypan blue exclusion test was> 96%. Cell culture Non-adherent mononuclear marrow cells were seeded in 12-well tissue culture plates containing RPM1 1640 medium with 20% heat-inactivated (56OC for 30 min) fetal calf serum and 10% 5637-conditioned medium which was prepared as described before [21], at a concentration of 5 x lo5 cells/ml with 160 mM DMSO, 100 mM DMF or 2 mM hexamethylene bisacetamide (higher concentrations of the 3 polar compounds were cytotoxic and might kill immature cells selectively) and without as a control and incubated for 6 days at 37 OC in a humidified incubator under 5% co*. Assessment of differentiation at day 6 Morphologically, using Hyel-Romanowsky stain, the cytocentrifuge smears were prepared on day 6 of culture and at least 200 cells were scored for morphological maturation. Nitroblue tetrazolium due reduction test was carried out as described before [14] and at least 200 cells were scored for the presence of intracellular blue-black formazan granules. Results
The incubation of normal non-adherent mononuclear marrow cells with the 3 polar compounds for the entire 6 days of culture did not affect their survival (Table 1). A genuine differentiation had taken place with an absolute and not only proportional increase in both morphologically mature cells and nitroblue tetrazolium dye reducing cells (Table 1). The increase represents a true stimulation of differentiation rather than a selective killing of the immature cells since there was an increase in the total number of viable cells at day 6 compared to those cultured at day 0 (Table 1). The addition of 160 mM DMSO to cultures caused an increase of 13.5-15.2% in the number of mature granulocytes and a decrease in
l l
216 112 132 159
f f f f
10.4 18.0 18.1 14.0
Blasts, promyelocytes and myelocytes
Morphology’ l
395 318 302 336
f f f f
14.6 16.7 12.2 18.2
Metamyelocytes
(Absolute no. x 105)
229 260 244 264
f f f +
10.6 14.9 14.9 18.4
Mature Granulocytes
17 17 17 18
f f f k
1.9 1.7 1.8 1.6
Monocytes
on the differentiation of human normal myeloid immature marrow cells.
DMSO: dimethylsulphoxide, DMF: dimethylformamide, HMBA: hexamethylene bisacetamide. ‘In control and treated cultures after six days of incubation. The initial cell number on day 0 was 5 x 105. l ‘Differential count of 500 cells counted on cytocentrifuge smears of control and treated cultures on day 6. ***Mean f S.D. (n = 22).
1.8’ 1.4 1.8 1.6
8.5 7.0 6.8 7.5
Control 160 mM DMSO 100 mM DMF 2 mM HMBA
f f + f
Number of viable cells (x 105)*
Effect of Polar-planar compounds
Polar-planar compound
Table 1.
627 774 727 786
zt f f f
10.6 17.8 14.5 16.4
Nitroblue tetrazolium dye reducing cells (Absolute no. x 105)
40
immature cells of 39.5-52% compared with control cultures. The number of monocytes was not changed (Table 1). After incubation with 160 mM DMSO for 6 days, the nitroblue tetrazolium due reducing cells increased by 23.5-29.796 compared with control cultures. The addition of 100 mM DMF to cultures caused an increase of 6.7-12.646 in the number of mature granulocytes and a decrease in immature cells of 36.3-41.2% compared with control cultures. The number of monocytes was not changed (Table 1). After incubation with 100 mM DMF for 6 days, the nitroblue tetrazolium dye reducing cells increased by 13.5-15.9% compared with control cultures. The addition of 2 mM hexamethylene bisacetamide (HMBA) to cultures caused an increase of 12.6-15.1% in the number of mature granulocytes and a decrease in immature cells of 21.1-26.3% compared with control cultures. The number of monocytes was not changed (Table 1). After incubation for 6 days with 2 mM HMBA, the nitroblue tetrazolium dye reducing cells increased by 22.1-25.4% compared with control cultures. Most of the metamyelocytes and all mature granulocytes reacted positively with nitroblue tetrazolium dye. The exposure of normal human immature marrow cells to 160 mM DMSO for 6 days in culture reduced the immature cell population to 52 46 of control cultures. DMF (100 mM) and HMBA (2 mM) caused less decline of the immature cell population to 61.2% and 73.7%, respectively, of control cultures. The decline was associated with an increase of mature granulocytes after 6 days of culture with 160 mM DMSO, 100 mM DMF and 2 mM HMBA to 113.5%, 106.7% and 115 % of control cultures, respectively. Discussion
Effective control of neoplastic growth could depend upon the ability to manipulate tumour heterogeneity. Methods of directing and controlling the differentiation of tumour cells could potentially limit heterogeneity through the
conversion of tumour subpopulations into a single more differentiated phenotype [9]. Such single phenotype could be an easier target for conventional cytotoxic therapy [5]. Exposure of malignant cells to polar compounds followed by treatment with cytotoxic agents has been reported to enhance their antitumour effectiveness [ 1,4,7,18]. This simplifies therapy as less dosage and/or duration of cytotoxic drugs would be required to treat such homogenized tumour which can respond in a more uniform fashion. The membrane changes caused by polar compounds may facilitate the transport of cytotoxic drugs into the malignant cells. Also, the alterations in enzyme activities which are important in drug metabolism could make the primed tumour more responsive to the appropriate cytotoxic drug [24]. In the present study, the results showed that the 3 polar compounds: DMSO, DMF and HMBA are able to increase the number of normal human myeloid mature marrow cells and decrease the immature myeloid marrow cells after 6 days of culture. While a decrease of immature marrow cells to 52% of control in DMSO-treated cultures was associated with only 113.5% increase of mature cells, in HMBA-treated cultures a less decrease of immature marrow cells to only 73.7% of control was associated with 115% increase of mature cells. Apart from the hepatic, ocular and nervous toxicities of polar compounds, these results may indicate that long-term exposure to them could lead to a decrease in the population of normal human myeloid immature marrow cells in the order DMSO > DMF > HMBA. Although, phase I clinical trials of polar compounds have not yet shown any myelosuppression [3,6,8,19,22], the results of the present study together with those reported by Wiemann et al. [25] anticipate a premature senescence of normal immature marrow cells following long-term exposure to these agents. A novel approach to control malignant diseases would be achieved by the development of less toxic and more effective analogues of polar compounds for clinical trials [ 15,161.
41
References 1
2
3
4
5
6
7
8
9
10
11
12
Arundel, CM., Glkksman. A.S. and Leith, J.T. (1985) In vitro effects of N-N-dimethylformamide on sublethal and potenttally lethal damage recovery processes after X-radiation in heterogeneous human colon tumour cells. Cancer Res., 45,5557-5562. Calabresi, P., Dexter, D.L. and Heppner, G.H. (1979) Clinical and pharmacologic implications of cancer cell differentiation and heterogeneity. B&hem. Pharmacol., 28, 1933- 1939. Chun, H.G., Leyland-Jones, B., Hoth, D., Shoemaker, D., Wolpert-DeFillippes, M., Grieshaber, C., Cradock, J., Davidgnon, P., Moon, R., Rifktnd, R. and Wittes, R.E. (1986) Hexamethylene bisacetamide: A polar-planar compound entering clinical trials as a differentiating agent. Cancer Treat. Rep., 70,991-996. Dexter, D.L., DeFusco, D.L. and McCarthy, K. (1983) Polar solvents increase the sensitivity of cultured human colon cancer cells to &-platinum and mitomycin C. Proc. Am. Assoc. Cancer Res., 24,267. Dexter, D.L., Leith, J.T. and Crabtree, G.W. (1982) N-Ndimethylformamide induced modulation of responses of tumour cells to conventional anticancer treatment modalities. In: Maturational factors and Cancer, pp. 105112. Editor: M.A.S. Moore. Raven Press, New York. Egorin, M. J., Sigman, L.M., VanEcho, D.A., Forrest, A., Whitacre, M.Y. and Aiiner, J. (1987) Phase I clinical and pharmacological study of hexamethylene biiacetamide (NSC 95580) administrated as a five day continuous infusion. Cancer Res., 47,617-623. Einspenner, M., Boulton, J.E. and Borsa, J. (1984) Variation of radiation sensitivity of friend erythroleukaemia cells cultured in the presence of the differentiation inducer: DMSO. Rad. Res., 97.55-63. Ettinger, D.S., Grr, D.W., Rice, A.P. and Donehower, R.C. (1985) Phase I study of N-Methylformamide in patients with advanced cancer. Cancer Treat. Rep., 69, 489-593. Francis, G.E. and Pinsky, CM. (1987) Growth and differentiation control. In: Cancer chemotherapy and Biologtcal response modifiers, pp. 502-538. Editors: H.M. Pinedo, D.L. Longo and B.A. Chabner. Elsevter, Amsterdam. Hassan, H.T. (1988) Differentiation inducers of human myeloid leukaemia cell lines. Folia Haematol., 115, 887896. Hassan, H.T. and Rees, J.K.H. (1988) Effect of AML serum on the differentiation of normal myeloid progenitors and the hexamethylene b&a&amide-induced granulocyttc differentiation of the human HL-60 promyelocytic leukaemia cell line. Blood, 72,204a, 725. Hassan, H.T. (1988) Dtfferentiation induction therapy of
13
14
15
16
17
18
19
20 21
22
23
24
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