Cancer Letters, 51(1990)

209

209-212

Elsevier Scientific Publishers Ireland Ltd.

Morphological alterations induced by doxorubicin in Bl6 melanoma cells M. Mariani and R. Supino Division

of Experimental Oncology B, Istituto Nozionole Tumori, Vio Venezian 1 - 20133 Milan (Italy)

(Received 23 November 1989) (Accepted 15 February 1990)

Summary In Bl6 melanoma, cells morphologically different can be distinguished. fn order to estabcorrelations between cell lish possible morphology and drug-response, the cytotoxic response to doxorubicin was analyzed. The

two subpopulations, represented by two types of colonies, showed a different degree of sensitioity to doxorubicin. Moreooer, following treatment, colonies strongly altered in their morphology were found, suggesting a differentiating actiuity of doxorubicin (denditric prolongations, increase of intracellular melanin, block of cell proliferation). These results suggest that doxorubicin, besides haoing a different cytotoxic effect on the two cell subpopulations, induces in this cell line morphological alterations consistent with a differentiation process.

Keywords: differentiation;

melanoma;

doxo-

rubicin . Introduction

Tumor heterogeneity, i .e . , the coexistence of multiple cell subpopulations within the same tumor, is a possible explanation for the failure Correspondence to: R. Supino. ‘To whom requests should be addressed at the lnstituto Nazionale Tumori - Via Venezian 1,20133 Milan, Italy.

of cancer chemotherapeutic treatment [ 1,6]. This heterogeneity can be revealed as multiple cellular differences by morphological, biological and/or biochemical parameters; some of these differences could be related to the expression of drug resistance phenotypes contributing to the lack of response to the treatment [2,3]. Previously [lo] we reported that in B16V, a murine melanoma cultured in vitro, 20% cells survived treatment with doxorubicin (DX) for 72 h at doses ranging between 60 ng/ml and 600 ng/ml; then it seems that a well-defined fraction of the cellular population is more resistant to DX than the cell majority. Furthermore, morphological observation of B 16V melanoma cells, revealed two cell populations: one of bipolar quite lengthened cells and the other of polygonal-shaped ceils with a few short dendritic processes. The aim of this study was to investigate possible correlations between cell morphology and doxorubicin response in this cell line, also taking into account some alterations related to differentiation such as cell growth and morphology and melanin content. A differentiating effect of DX similar to that of other antineoplastic drugs on different cell lines is assumed [5,8,9,12]. Materials and methods B16V cells were grown in RPM1 1640 +

210

Fe(CN),K 0.03 mM + 10% foetal calf serum (Flow La %oratories, Irvine, Ayrshire, U.K.) [lOI. For colony experiments, cells were seeded 2 x lo5 cell/plate of 5 cm cp. After 24 h cells were treated with DX at different concentrations for 24 h; then cells were washed and reseeded at 200 cells/plate. After about 1

week the colony shape and number were determined on plates fixed and coloured with crystal violet. To determine the cell shape, single cell suspensions were seeded in 96-well tissue culture plates (Costar, Cambridge, MS, U.S.A.) at a concentration of 1 cell/well. Wells with more than 1 cell were not taken into account. Growing colonies from each cell were observed in their dimensions and shape for a few days. Seven days after the cell seeding, when colony shape was defined, DX for different times and at different concentrations was added to each well. Cell and colony morphology was observed for a few days. Results and discussion

Two types of colonies were derived from B16V cells, reflecting the presence of two populations existing in the melanoma cell line: one consisted of compact cells bipolar or round (being the latter problably diving bipolar cells that round up) ; the second consisted of scattered polygonal cells with few intercellular contacts (Fig. 1). The colonies with sparse cells represented about 30% of the total colony number. Very few melanotic cells (approx. 2%) could be observed in both types of colonies and melanotic colonies were not found.

Table 1. B16V.

Doxorubicin activity on different colonies of

Doxorubicin h/ml)

200 40 8 1.6 0.32

ID, h/ml) Fig. 1. B16V colonies: (a) a compact colony (425 and (b) a scattered colony (425 X ) .

X)

No. of Colonies (% of control)” Compact

Scattered

0 0 5 35 80

0 6 25 97 100

0.9

4.6

aIn control, untreated cells compact colonies were 125 and scattered colonies were 51.

Fig. 2. A B16V cell after DX treatment: prolongations are evident (850 x )

dendritic-like

To evaluate the cytotoxic effect of DX on the two different colonies, cells were exposed to the drug for 24 h. Colonies with different morphology were counted and the results are shown in Table 1. It appears that compact colonies were more sensitive to DX having an ID,, of 0.9 ng/ml, while on the scattered colonies the ID,, was 4.6 ng/ml. As an increase of melanotic and dendriticlike cells was observed after drug treatment (Fig. 2) , some experiments were performed on colonies grown singularly in 96-well tissue culture plates (see Materials and methods) in order to determine whether this cell morphology modulation could be attributed to drug exposure. As reported in Table 2, DX I pg/ml for 48 h induced a cell death of 86% of coloTable 2.

Doxorubicin-activity

Fig. 3. Melanotic colonies observed concentration treatment (850 X )

after

DX high

on isolated B16V colonies.

DX concentration X time

Surviving at treatment

Alive 4 days after treatment

Alive 2 weeks after treatment

0.1 pg/ml x 48 h 1 &ml x 48 h lOO&ml x 24 h

80 14 26

80 8 16

80 0 2.2

212

nies; of the remaining 14% only 8% grew for a few days again but when trypsinized and passed they did not adhere to the culture plate. Surprisingly, after treatment with Dx 100 pg/ ml for 24 h, 26% of the round and melanotic cells were alive for 3 or 4 days: however, some of them lost their anchorage capability and stayed as single floating cells. In 2.2% of wells, round or dendritic highly melanotic cells, strongly anchored to the plastic substrate, were found (Fig. 3); these colonies remained alive for at least 10 weeks without cell replication. In conclusion, our results proved the presence of two cell subpopulations in B16V cell line. These cells are different with regard to cell morphology, shape of derived colonies and drug sensitivity being 5-fold higher in compact than in scattered colonies (Table 1 and Fig. 2). In addition high DX levels induce a cell proliferation block of a small percentage of cells without cell death, together with a strong increase in melanin intracellular content. In few cells dendritic-like processes appeared from the cell body with terminal granules. Although it is known that cell density can modulate the cell shape [ll], in this case the presence of melanotic and dendritic-like cells cannot be due to the low cell number as cells with a similar behaviour were never observed in untreated control low density cultures. Since it has been reported that the above examined parameters are indeces of cell differentiation [4,7,8], these results support that DX enhances alterations consistent with the cell maturation inducing a progress toward a terminal differentiation in cells selected for a higher drug resistance, besides inducing a selection of more resistant cells.

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