CELL BIOCHEMISTRY AND FUNCTION
VOL.
10: 9-17 (1992)
Identification of Multi-Drug Resistant Cells in Sensitive Friend Leukemia Cells by Quantitative Videomicrofluorimetry S. LAHMY,? J. M. SALMON, J. VIGO AND P. VIALLET Quantitative et Pharmacocint?iique Cellulaire' U R A CNRS 1289, Universitt. de Perpignan, 52 avenue de Villeneuve, 66860 PERPIGNAN Cedex ' Microfiuorirn6trie
The cellular resistance to cytotoxic drugs, particularly to anthracyclines, remains a major problem in cancer chemotherapy. A number of biochemical mechanisms have been described, one of them being a lower accumulation of drugs in resistant cells. The accumulation of Ho33342 in sensitive and resistant Friend leukemia cells was studied by quantitative fluorescence image analysis, a method which allows investigations to be made on living tissues and cells. The intensity of fluorescence is related to the amount of Ho33342 accumulated into the cells and has been found to be more intense in sensitive cells than in resistant ones. Moreover, the retention of this vital dye was inversely related to the degree of resistance in the three resistant cell lines. The addition of verapamil, which is known to reverse resistance to anthracyclines, resulted in an increase of the amount of Ho33342 accumulated in the resistant cells. Ho33342 presents a higher quantum yield than any other anthracyclines, such as adriamycin and can be used as a microfluorimetric probe to identify the resistant cells in a heterogeneous cell population. K F Y WORDS
-Hoechst 33342; Friend leukemia cells; multi-drug resistance, quantitative videomicrofluorimetry
INTRODUCTION During the treatment of tumours, resistance very frequently develops against the cytotoxic agent used. Treatment with other cytotoxic agents often fails, because the tumour cells exhibit cross-resistance not only to similar drugs, but also to drugs which are functionally and structurally unrelated. Multi-drug resistance (MDR) has been correlated with a low accumulation of anthracyclines in the nucleus'** and of rhodamines in the m i t ~ c h o n d r i a . ~The . ~ low accumulation of these compounds has been explained by an increased efflux in the resistant cells. In the main, two kinds of metabolic processes have been suspected to be responsible for this efflux: an energy-dependant efflux pump related to P-glycoprotein,'X6 which reduces intracellular concentrations of cytotoxic drugs;'^^ and an increase in gluthatione transferase activity which in turn would activate the cellular detoxification system (increase in the glutathione conjugation of the drugs), leading to elimination of the cytotoxic Finally, the resistance can be reversed by the use of substances such as verapamil, a calcium channel blocker, which increases the t Address [or correspondence 0263 6484/92/01ooO9- 17$08.50 0 1992 by John Wiley & Sons, Ltd.
sensitivity of the resistant cells by preventing the drug efflux.'"3 Various studies on drug resistance have been carried out by flow cytometry.'"'6 This method, like any other optical method is non-destructive and allows living material to be studied, especially single living cells. Fluorescence image analysis, another non-destructive procedure, has been compared with flow cytometry for studies on intact cells. Because cells can be observed for a longer period than in flow cytometry, additional information can be obtained from the same cell including cellular distribution of probes and information about size, shape, and fluorescence intensity. Furthermore, this method allows cells, both in suspension and monolayers, to be studied without being treated, e.g. with trypsin, to detach the cells. Therefore this technique seems an attractive tool for the study of single living cells. Fluorescence image analysis has been used to detect the resistant cells in a heterogeneous mixture. As a first step t o distinguish resistant cells, the accumulation of adriamycin (ADR) in sensitive Friend leukemia cells and Friend leukemia cells presenting three levels of resistance to ADR was studied. During this study, the cell cycle was monitored with the vital stain Hoechst 33342, which
10 binds specifically to A-T sequences of DNA,” to determine whether there was a correlation between nuclear accumulation of ADR and the phases of the cell cycle. This experiment showed that more Ho33342 accumulates in the sensitive cells than in the resistant ones. Lalande et a l l 8 also reported similar results with respect to accumulation of Ho33342. It has already been demonstrated” that the fluorescence quantum yield of Ho33342 bound to DNA is 100 times greater than that of the free dye. In contrast, the fluorescence quantum yield of ADR is five-fold less when bound to DNA.,, A spectrofluorometric study of Ho33342 and ADR bound to DNA gave a quantum yield for Ho33342 10 times higher than for ADR (unpublished results). Ho33342 is also a vital stain which avoids problems of toxicity. Thus it seems that Ho33342 is a more appropriate probe than ADR for the detection of resistant cells, owing to its higher signal/noise ratio in cells and the lack of toxicity. However, because a spectral shift of this probe has been used to discriminate resistant cells from sensitive ones,21 first a spectral study by microspectrofluorometry was carried out, and the fluorescence spectra of Ho33342 accumulated into nuclei were compared with those for Ho33342 bound to DNA. A protocol designed to achieve easy and reliable detection of cellular resistance to cytotoxic drugs by means of fluorescence image analysis of Ho33342 was explored. Initially, incubation times and concentrations required for the accurate evaluation of the intracellular accumulation of the probe into cells were established. Results obtained for the sensitive cells and three cell lines derived from these sensitive cells, which have different degrees of resistance to ADR are presented. The effect of verapamil, which is known to reverse multi-drug resistance, was also tested on one of the resistant lines (RFLC3). The protocol was then applied to a mixture of RFLC3 and sensitive cells, to determine the reliability of the method. MATERIALS AND METHODS Cells
Friend leukemia cells sensitive (FLC sens) and resistant to ADR (RFLC1, RFLC2, RFLC3) were kindly provided by Dr H. Tapiero (ICIG, Villejuif, France). The resistant cells were derived from the sensitive ones by continuous exposure to ADR, as
S. LAHMY ET A L .
described by Tapiero et ~ l . RFLC3 , ~ being the most resistant ones. Once resistance was acquired, the cells were grown in a drug-free medium. The Gl,, (50 per cent inhibition of growth) for RFLCl, RFLC2 and RFCL3 were 20ng ml-’, 400ng ml-’, and 1300ng m1-l. They were cultivated routinely in RPMI 1640 (Flow) supplemented with 2 mM glutamine (Flow), 10 per cent of decomplemented foetal calf serum (Gibco), and antibiotics. All cell cultures were grown at 37°C in a CO, incubator. To maintain continuous exponential growth, cells were seeded at 0.1 x lo6 cells mland passaged every 2 or 3 days. All experiments were performed with cells in the exponential growth phase. In all experiments, the viability of the cells was measured by counting cells excluding 0.1 per cent trypan blue, and this was never below 95 per cent. The cell lines were tested regularly and found to be mycoplasma-free.
’
Exposure to Ho 33342
The chemicals used were all of analytical grade. Ho33342 (2’-(4-ethoxyphenyl)-5-(4-methyl1-piperazinyl)-2,5’-bi-l H-benzimidazole) from Aldrich was dissolved in phosphate-buffered saline (PBS) at l o p 3 mole per liter. The cell suspension (1 ml) was added to an aliquot of the stock solution. The final concentration of Ho33342 was 1 0 p in ~ the culture medium except where otherwise mentioned. Cells were incubated for 2 h at 37°C and then centrifuged (3 min, 100 y). They were rinsed three times with cold PBS (4°C). We then examined the cells in Sykes-Moore chambers without the upper lamella by image analysis. When necessary, verapamil from Sigma (10 PM, final concentration) was added to the cell suspensions 30min before the addition of Ho33342, and this was maintained during the incubation and rinsing. Fluorescence Spectra of Stained Nuclei
We recorded the fluorescence spectra of Ho33342 bound to DNA in solution, or accumulated in the nuclei of the sensitive and resistant cells using a microspectrofluorimeter built in our l a b ~ r a t o r y . ~The ~ . ~excitation ~ wavelength was 340 nm. Numerical Image Analysis
The number of sampled nuclei in each distribution was in the range of 300-350. The videomicro-
11
VIDEOMICROFLUORIMETRY OF MDR CELLS
Ruorimetry system has already been described.24 It consists of an inverted fluorescence microscope (Olympus, IMT2) equipped with an epi-illuminating system, a 40 x objective (Leitz), and a S.I.T. (Silicon Intensified Target) camera from Lhesa coupled to a TITN SAMBA 2002 image processor (see Figure 1). Depending on the excitation wavelength required, a mercury or xenon lamp can be used. For these experiments, a dichroic mirror selected the line 365 nm of a high-pressure mercury lamp (100 W) for fluorescence excitation. Quantitative measurements were possible, since a special data acquisition program was developed to avoid artefact^.'^ Precautions include the removal of the background, the correction of the non-linearity of the digitizer and the heterogeneity of the camera gain. pixel to pixel. This was made possible by using an homogeneous fluorescent reference sample for all experiments. Furthermore, this reference sample enabled us to compare one experiment with another.25Since Ho33342 is fluorescent only when bound to DNA, the fluorescence signal came from the nucleus. A protocol to record only the nuclear fluorescence was developed: image segmentation by thresholding, followed by a cell contour smoothing by opening and closing sequences. The use of parameters relative to the surface of the nuclei and
t
Figure 1.
Scheme of the videomicrofluorimetry apparatus.
to the fluorescence intensity makes rejection of subcellular debris, or cell clumps possible. RESULTS Staining Conditions
In every case the fluorescence properties of a probe must be studied and analyzed before quantification. Therefore a spectroscopic study of the fluorescence of stained nuclei has been performed. There was no apparent difference between the fluorescence spectra of the sensitive cells and those of the resistant ones. As shown in Figure2, there was no difference between the fluorescence spectrum of Ho33342 bound to DNA in solution or accumulated in the nuclei of resistant and sensitive cells. Thus it was assumed that the interactions between Ho33342 and its environment were similar whether Ho33342 was bound to DNA or accumulated in the cells. The conditions of quantitative measurements of Ho33342 fluorescence were next defined. In order to determine the concentrations to be used, two different concentrations of the probe 5 0 p ~and
12
S. LAHMY ET AL.
IF counts
i
400
500
600
h (nm)
Figure 2. Comparison of fluorescence spectra for sensitive (0) and resistant ( 0 )cells after staining with Ho33342 (10 PM) and Ho33342 bound to DNA in solution (.). Fluorescence spectra have been normalized to show the absence of shift. The spectrum of resistant cells has been multiplied by 4.
10 ,UM, were tested on the sensitive and the resistant cells. Figure 3 shows the distributions of fluorescence intensity for both concentrations of Ho33342 obtained for the sensitive cells (A,B) and the resistant cells (C,D). Comparison of the two doses on the sensitive cells showed that (1) after staining with the probe at 10 PM, a classical histogramz6 was observed with a peak corresponding to cells in G0/1 phase at 15000 counts and a peak corresponding to cells in G2 + M phase at 30000 counts; (2) after staining with the probe at 50 ,UM, unexpected results were obtained: the fluorescence intensities were in the same range of values as those obtained after staining at 10 ,UM although higher fluorescence intensities would have been expected. Furthermore, the distribution of fluorescence intensities was slightly shifted towards lower values. This could be explained by fluorescence quenching due to a high concentration locally. In the case of the resistant cells, no significant difference was observed between the two concentrations, but the resulting histograms were completely different from those for sensitive FLC: lower fluorescence intensities were obtained, as well as different cell distributions. However, the use of ethidium bromide has shown no difference in DNA content between the sensitive and the resistant cells.27Thus it was concluded that Ho33342 cannot
be used as an indicator for the cell cycle in the resistant cells. Therefore the choice was made to perform the experiments with 1 0 , to~ avoid ~ any perturbations or variations due to the concentration of the probe. Because staining time is a major variable, variation of nuclear fluorescence intensity as a function of incubation time was also studied. Both curves in Figure 4 show that after 2 h of incubation, staining reached a plateau and that there were no significant variations in the staining thereafter. Consequently, this period of incubation was chosen for the experiments on the sensitive and the resistant cells. These conditions allowed comparisons to be made between the fluorescence intensities of the cell populations because the staining conditions were equivalent. These curves also indicated that the fluorescence intensity (i.e. the amount of probe accumulated in the nucleus) was lower in the resistant cells than in the sensitive ones.
Accumulation of Ho33342 in Sensitive and Resistant Cells Having established the staining conditions, the accumulation of Ho33342 was studied in the different cell lines, all observed in their exponential growth phase: FLC sens, RFLC1, RFLC2 and
13
VIDEOMICROFLUORIMETRY OF M D R CELLS
t
t C
D
"'i
23.3
11.7
5.7
12000
IF
36000
12000
36000
Figure 3. Influence of drug concentrations in the staining. Cell distribution (%) as a function of fluorescence intensity 1a.u.) Sensitive--A: 10 pM, B: 50 L ~ MRFLC3-C: . 10 pM, D: 50 pM. I50000
T 5
100000
Ci
+.
.-*m
-
I
/
C
QJ
5
i
50000
I
1
0
0
1
2
3
4
5
6
hours Figure 4. Influence of incubation time for the staining of sensitive ( 0 )and resistant cells (+).
RFLC3 (Figure 5). The first peak of the distributions was used to compare the cell populations. For RFLC1, the distribution showed lower intensity values and a shift in the peak value of - 20 per cent compared to the peak obtained with
the sensitive cells. This shift increased for RFLC2 and reached -73 per cent for the peak of RFLC3. This represented a factor of 4 in the level of Ho accumulation between FLC sens and RFLC3. For clarity, the position of the peak of sensitive cell distribution is indicated by a dotted line in the four distributions. These results show that the accumulation of Ho33342 is inversely correlated to the resistance level. Figure 5 also shows that the values of fluorescence intensity for RFLCl were in the same range as the sensitive cells, but that there was a slight shift towards low values. The peak position in the distributions of RFLC2 and RFLC3 was completely shifted, and out of the range of the distribution of the sensitive cells. Nevertheless, some cells exhibited fluorescence intensities in the range of those of the sensitive cells. The number of these cells decreased with increase in cell resistance. Efeect of Verupumil
To test the relationship between low nuclear accumulation of Ho33342 and cellular resistance to ADR, we tested the effect of varapamil (VPL) on resistant cells (RFLC2, RFLC3). VPL is a calcium channel blocker and is often used to reverse multi-
14
S. LAHMY ET A L .
25.8
RFLC2
5.2 W
v
$?
A
12.8
RFLC3
7.7
2.6
6.9
0
12000
36000
IF
12000
36000
IF
) sensitive and resistant cells. Cell distributions (%) as a Figure 5 . Differences in accumulation of Ho33342 (10 p ~ for function of fluorescence intensity (a.u.). Dotted line indicates the peak of sensitive cells.
drug Figure 6 shows the distributions in the presence and absence of VPL for RFLC3. In the absence of VPL, the mean value of the population located under 12000 counts was 4741 with a standard deviation of 1642 counts. After addition of VPL, the mean value of the cell population under 12000 counts was 6670 with a standard deviation of 2192 counts. This shift represents an increase of 30 per cent of the fluorescence intensity: according to the Fisher-Snedecor test, the populations are different at 99 per cent.28 Similar results were found for RFLC2 (data not shown). However, in neither case did the intensities of fluorescence with VPL treatment reach the values obtained with the sensitive cells. This agrees with the findings of Morgan et aL2' Addition of VPL, at the same concentration to sensitive cells had no effect on the accumulation of Ho33342.
RFLC3
Deiection of Resistant Cells in a Mixture
To check the potential of this method, it was applied to the detection of resistant cells in a mixture of cells. Figure 5 shows that it would be difficult to detect t i e percentage of RFLCl in a
12000 36000 IF Figure 6. Effect of verapamil (10 PM) on the accumulation of Ho33342 ( 1 0 ~in~ RFLC3. )
VIDEOMICROFLUORIMETRY OF MDR CELLS
mixture of sensitive and RFLCl cells. For this reason a study was carried out on a mixture of RFLC3 and sensitive FLC. RFLC3 and sensitive cells were mixed at the same density in a ratio of 1 :2. Cells were incubated according to the procedure described in ‘Materials and methods’. The histogram of fluorescence intensities of the mixture is shown in Figure 7. Two groups of cells would be distinguished. The first had a value with a maximum at 4000 counts and represented about 35 per cent of the whole population. The second had its maximum at 16000 counts and represented 65 per cent of the cells. There was a factor 4 between the two peak values, which corresponds to that observed for the Ho33342 accumulation into the sensitive and the RFLC3. A similar experiment was also carried out with a mixture and the RFLC2 and sensitive cells (data not shown). Although it was possible to distinguish cells with a high resistance level, these measurements show that the method is not accurate enough for the cells with a low resistance level (namely RFLC1). DISCUSSION Different approaches have been used to detect the resistance of tumour cells to cytotoxic drugs. These include the presence of P-glycoprotein duc to MDR e x p r e s ~ i o n ; increased ~,~ glutathione transferase a ~ t i v i t y ; ’ ~ .and ~ ’ nuclear accumulation of fluorescent cytotoxic drugs.’,16 The disadvantage associated with the last method is the low quantum yield of the compounds, particularly ADR, when
12000
24000
IF
Figure 7. Distribution of fluorescence intensities for a mixture of sensitive and RFLC3 cells.
15
bound to DNA. Thus, for accurate measurements, high concentrations of the drugs have to be used in investigations on single living cells, but then it becomes difficult to distinguish toxic effects from cellular events. The advantages of the Ho33342 are that not only can low concentrations of the probe (10 j t instead ~ of 100 PM”) be used due to its high quantum yield of fluorescence, but also this vital stain does not affect living cells. However, in observations at 400 and 600 nm by flow cytometry, Morgan et al.” mentioned spectral shifts in the spectra of Ho33342 accumulated in the nuclei of sensitive and resistant small lung cells. We have therefore studied the fluorescence spectra of nuclear Ho33342 accumulated in sensitive and resistant FLC, before any comparison of fluorescence intensities by numerical image analysis were made. No differences were observed between these fluorescence spectra and the fluorescence spectrum of Ho33342 bound to DNA. This indicates that the molecular environment of the probe is similar in both cases. The results obtained indicate that the conditions described for the staining represent a convenient protocol. Comparison of the histograms of fluorescence intensities for RFLC1, RFLC2, and RFLC3 have shown (1) substantial differences between fluorescence intensities of the sensitive cells, RFLC2 (GIso = 400ng ml-’) and RFLC3 (GIso = 1300 ng ml- I); (2) only slight differences between sensitive cells and RFLCl (GI,, = 20 ng ml-’); (3) that Ho33342 cannot be used to monitor phases of the cell cycle in resistant cell lines, since accumulation of the probe depends on the resistance level. However, there was no difference in the DNA content between sensitive and resistant cells.27 The treatment of RFLC3 and RFLC2 by VPL, known as a resistance modulator, led to a higher accumulation of Ho33342. This indicates that the nuclear accumulation of this probe can be related to the resistance. We can thus use it as a marker for resistance, and the potentialities of this probe have been confirmed by the detection of resistant cells in a mixture of sensitive cells and RFLC3. The protocol should now be improved to determine the percentage of resistant cells in a mixture with better accuracy. Although Ho33342 appears to be a suitable probe to discriminate resistant cells with quite a high level of resistance (GIso 2 400 ng ml- l), the results emphasize the difficulties of discriminating the low level resistant cells (GI5o I 20 ng ml-I).
16
In future studies, Ho33342 will be used to follow the reversion of the resistance in RFLC2 and RFLC3, following addition of other modulators. The relationships between resistance and other cellular parameters such as pH, C a 2 + , Mg2+ and mitochondria1 rhodamine accumulation, will be studied using Ho33342 as the resistance indicator. Secondly, we will study the possibility of using Ho33342 for detection of cells with low resistance level. We therefore need to determine the lowest difference in nuclear fluorescence which will allow a reliable distinction between resistant and sensitive cells. This may be done by growing cells in a medium containing ADR (which induces resistance) and by simultaneously evaluating the amount of Ho33342 accumulated in the cells. ACKNOWLEDGEMENTS We are indebted to Professors E. Kohen and J. Hirschberg for their critical readings. We thank Dr Tapiero and Mrs Trincal for providing the cells and FrCdkric Bancel for helpful discussions. This work was supported by INSERM CRE NO899015 and by the ‘Ligue Francaise contre le cancer’, and the ‘P81e regional Languedoc Roussillon de Genie Biologique et Mkdical’. REFERENCES 1. Skovsgaard, T. and Nissen, N. I. (1982). Membrane transport of anthracyclines. Pharmac. Ther., 18, 293-31 1 . 2. Fojo, A., Shin-ichi, A., Gottesman, M. M. and Pastan, I. (1985). Reduced drug accumulation in multiple drugresistant human KB carcinoma cell lines. Cancer Res., 45, 3002-3007. 3. Lampidis, T. J., Bernal, S. D., Summerhayes, I. C. and Chen, L. B. (1982). Rhodamine 123 is selectively toxic and preferentially retained in carcinoma cells in uitro. Ann. N Y Acad. Sci., 99, 299-302. 4. Tapiero, H., Munck, J. N., Fourcade, A. and Lampidis, T. J. (1984). Cross resistance to rhodamine 123 in adriamycin and daunorubicin resistant Friend leukemia cell variants. Cancer Rex, 44,5544-5549. 5. Willingham, M. C., Richert, N. D., Cornwell, M. M., Tsuruo,T., Hamada, H., Gottesman, M. M. and Pastan, I. H. (1987). Immunocytochemical localization of PI70 at the plasma membrane of multidrug-resistant human cells. J . Histochem. Cytochem., 35, 1451-1456. 6. Juranka, P. F., Zastawny, R. L. and Ling, V. (1989). Pglycoprotein: multi-drug resistance and a superfamily of membrane associated transport proteins. FASEB J., 3, 2583-2592. 7. Roninson,I. B.,Patel,M.C.,Lee,I.,Noonan,K. E.,Chen,C. J., Choi, K., Chin, J. E., Kaplan, R. and Tsuruo, T. (1989). Molecular mechanism and diagnostics of multidrug resistance in human tumor cells. Cancer Cells, 7, 81-86.
S. LAHMY ET AL. 8.
9.
10.
11. 12.
13.
14. 15. 16.
17.
18.
19. 20.
21.
22.
23.
Bradley, G.,Juranka, P. F. and Ling, V. (1988). Mechanism of multidrug resistance. Biochem. Biophys. Acfa, 948, 87-128. Moscow,J.A., Fairchild,C. R., Madden,M. J., Ransom,D.T, Wieand, H. S., O’Brien, E. E., Poplack, D. G., Cossman, J., Myers, C. E. and Cowan, K. H. (1989). Expression of anionic glutathione-S-transferase and P-glycoprotein genes in human tissues and tumors. Cancer Res., 49, 1422- 1428. Tew, K. (1989). The involvement of glutathione S-tranferases in drug resistance. In: Anticancer Drugs (Tapiero, H., Robert, J. and Lampidis, T., eds) Colloque INSERM/ John Libbey Eurotext, pp. 103-112. Lampidis, T. J., Krishan, A., Planas, L. and Tapiero, H. (1986). Reversal of intrinsic resistance to adriamycin in normal cells by verapamil. Cancer Drug Deliu., 3,25 1-259. Yusa, K. and Tsuruo, T. (1989). Reversal mechanism of multidrug resistance by verapamil: direct binding of verapamil to P-glycoprotein on specific sites and transport of verapamil outward across the plasma membrane of K562/ ADM cells. Cancer Rex, 49, 5002-5006. Willingham, M. C., Cornwell, M. M., Cardarelli, C. O., Gottesman, M. M. and Pastan, I. (1986). Single cell analysis of daunomycin uptake and efflux in multidrug resistant and sensitive K B cells: effects of verapamil and other drugs. Cancer Res., 46, 5941-5946. Hedley, D. W., Hallahan, A. R. and Tripp, E. H. (1990). Flow cytometric measurement of glutathione content of human cancer biopsies. Br. J. Cancer, 61, 65-68. Herweijer, H., Van den Engh, G. and Nooter, K. (1989). A rapid and sensitive flow cytometric method for the detection of multidrug resistant cells. Cyfomefry,10, 463-468. Krishan, A. and Ganapathi, R. (1979). Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry. J. Histochem. Cytochem., 21, 1655-1656. Erba. E.. Ubezio. P.. Bronnini. M.. Ponti. M. and D’Incalci. M. (1988). DNA damage, cytotoxic effect and cell cycle perturbation of Hoechst 33342 on L1210 cells in vitro. Cytomefry,9, 1-6. Lalande, M. E., Ling, V. and Miller, R. G . (1981). Hoechst 33342 dye uptake as a probe of membrane permeability changes in mammalian cells. Proc. Nufl. Acad. Sci. U S A . , 78, 363-367. Kapunscinski, J. (1990). Interactions of nucleic acids with fluorescent dyes: spectral properties of condensed complexes. J. Histochem. Cytochem., 38, 1323- 1329. Ginot, L., Jeannesson, P., Angiboust, J. F., Jardillier, J. C. and Manfait, M. (1989). Interactions of adriamycin in sensitive and resistant leukemia cells. A comparative study by microspectrofluorometry. Studiu Biophysica, 104, 145- 153. Morgan, S. A,, Watson, J. V., Twentyman, P. R. and Smith P. J. (1989). Flowcytometric analysis of Hoechst 33342 uptake as an indicator of multidrug resistance in human lung cancer. Br. J. Cancer, 60,282-287. Salmon, J. M., Vigo, J. and Viallet, P. (1981). Microspectrofluorimttrie sur cellules vivantes isoltes: couplage d’un microspectrofluorimetre a un microordinateur. Innoo. Tech. B i d . Med., 2, 679-686. Lahmy, S., Salmon, J. M. and Viallet, P. (1987). Effect of ellipticine on M F O activity in single living 3T3 fibroblasts studied by microspectrofluorimetry. Anticancer Res., 7, 353-360.
17
VIDEOMICROFLUORIMETRY OF MDR CELLS
24. Lahmy, S., Salmon, J. M., Vigo, J. and Viallet, P. (1989). pHi and DNA content modifications after ADR treatment in 3T3 fibroblasts. A microfluorimetric approach. Anficancer Rex, 9, 929-936. 25. Vigo, I., Salmon, J. M., Lahmy, S. and Viallet, P. (1991). Videomicrofluorimetry: from qualitative to quantitative measurements. Anal. Cell. Path., 3, 145- 165. 26. Arndt-Jovin, D. J. and Jovin, T. M. (1977). Analysis and sorting of living cells according to deoxyribonucleic acid content. J . Histoehem. Cyfochern.,25, 585-589. 27. Lahmy, S., Salmon, J. M., Vigo, J. and Viallet, P. (1991). Accumulation of adriamycin in Friend leukemia cells: image analysis applied to resistance mechanisms. J . Cell. PharmacoL, 2, 18-23.
28. Lamotte, M. (1967). In: Initiation aux MPthodes Stafistique.< Masson and Co: Paris, pp 76-86. 29. Lee, F. Y., Sciandra, J. and Siemann, D. W. (1989). A study of the mechanism of resistance to adriamycin in v i m . Biochem. Pharmaco/., 38, 3697-3705.
Received in revisedforni 29 July 1991 Accepted 2 September 1991
VOL.
10: 9-17 (1992)
Identification of Multi-Drug Resistant Cells in Sensitive Friend Leukemia Cells by Quantitative Videomicrofluorimetry S. LAHMY,? J. M. SALMON, J. VIGO AND P. VIALLET Quantitative et Pharmacocint?iique Cellulaire' U R A CNRS 1289, Universitt. de Perpignan, 52 avenue de Villeneuve, 66860 PERPIGNAN Cedex ' Microfiuorirn6trie
The cellular resistance to cytotoxic drugs, particularly to anthracyclines, remains a major problem in cancer chemotherapy. A number of biochemical mechanisms have been described, one of them being a lower accumulation of drugs in resistant cells. The accumulation of Ho33342 in sensitive and resistant Friend leukemia cells was studied by quantitative fluorescence image analysis, a method which allows investigations to be made on living tissues and cells. The intensity of fluorescence is related to the amount of Ho33342 accumulated into the cells and has been found to be more intense in sensitive cells than in resistant ones. Moreover, the retention of this vital dye was inversely related to the degree of resistance in the three resistant cell lines. The addition of verapamil, which is known to reverse resistance to anthracyclines, resulted in an increase of the amount of Ho33342 accumulated in the resistant cells. Ho33342 presents a higher quantum yield than any other anthracyclines, such as adriamycin and can be used as a microfluorimetric probe to identify the resistant cells in a heterogeneous cell population. K F Y WORDS
-Hoechst 33342; Friend leukemia cells; multi-drug resistance, quantitative videomicrofluorimetry
INTRODUCTION During the treatment of tumours, resistance very frequently develops against the cytotoxic agent used. Treatment with other cytotoxic agents often fails, because the tumour cells exhibit cross-resistance not only to similar drugs, but also to drugs which are functionally and structurally unrelated. Multi-drug resistance (MDR) has been correlated with a low accumulation of anthracyclines in the nucleus'** and of rhodamines in the m i t ~ c h o n d r i a . ~The . ~ low accumulation of these compounds has been explained by an increased efflux in the resistant cells. In the main, two kinds of metabolic processes have been suspected to be responsible for this efflux: an energy-dependant efflux pump related to P-glycoprotein,'X6 which reduces intracellular concentrations of cytotoxic drugs;'^^ and an increase in gluthatione transferase activity which in turn would activate the cellular detoxification system (increase in the glutathione conjugation of the drugs), leading to elimination of the cytotoxic Finally, the resistance can be reversed by the use of substances such as verapamil, a calcium channel blocker, which increases the t Address [or correspondence 0263 6484/92/01ooO9- 17$08.50 0 1992 by John Wiley & Sons, Ltd.
sensitivity of the resistant cells by preventing the drug efflux.'"3 Various studies on drug resistance have been carried out by flow cytometry.'"'6 This method, like any other optical method is non-destructive and allows living material to be studied, especially single living cells. Fluorescence image analysis, another non-destructive procedure, has been compared with flow cytometry for studies on intact cells. Because cells can be observed for a longer period than in flow cytometry, additional information can be obtained from the same cell including cellular distribution of probes and information about size, shape, and fluorescence intensity. Furthermore, this method allows cells, both in suspension and monolayers, to be studied without being treated, e.g. with trypsin, to detach the cells. Therefore this technique seems an attractive tool for the study of single living cells. Fluorescence image analysis has been used to detect the resistant cells in a heterogeneous mixture. As a first step t o distinguish resistant cells, the accumulation of adriamycin (ADR) in sensitive Friend leukemia cells and Friend leukemia cells presenting three levels of resistance to ADR was studied. During this study, the cell cycle was monitored with the vital stain Hoechst 33342, which
10 binds specifically to A-T sequences of DNA,” to determine whether there was a correlation between nuclear accumulation of ADR and the phases of the cell cycle. This experiment showed that more Ho33342 accumulates in the sensitive cells than in the resistant ones. Lalande et a l l 8 also reported similar results with respect to accumulation of Ho33342. It has already been demonstrated” that the fluorescence quantum yield of Ho33342 bound to DNA is 100 times greater than that of the free dye. In contrast, the fluorescence quantum yield of ADR is five-fold less when bound to DNA.,, A spectrofluorometric study of Ho33342 and ADR bound to DNA gave a quantum yield for Ho33342 10 times higher than for ADR (unpublished results). Ho33342 is also a vital stain which avoids problems of toxicity. Thus it seems that Ho33342 is a more appropriate probe than ADR for the detection of resistant cells, owing to its higher signal/noise ratio in cells and the lack of toxicity. However, because a spectral shift of this probe has been used to discriminate resistant cells from sensitive ones,21 first a spectral study by microspectrofluorometry was carried out, and the fluorescence spectra of Ho33342 accumulated into nuclei were compared with those for Ho33342 bound to DNA. A protocol designed to achieve easy and reliable detection of cellular resistance to cytotoxic drugs by means of fluorescence image analysis of Ho33342 was explored. Initially, incubation times and concentrations required for the accurate evaluation of the intracellular accumulation of the probe into cells were established. Results obtained for the sensitive cells and three cell lines derived from these sensitive cells, which have different degrees of resistance to ADR are presented. The effect of verapamil, which is known to reverse multi-drug resistance, was also tested on one of the resistant lines (RFLC3). The protocol was then applied to a mixture of RFLC3 and sensitive cells, to determine the reliability of the method. MATERIALS AND METHODS Cells
Friend leukemia cells sensitive (FLC sens) and resistant to ADR (RFLC1, RFLC2, RFLC3) were kindly provided by Dr H. Tapiero (ICIG, Villejuif, France). The resistant cells were derived from the sensitive ones by continuous exposure to ADR, as
S. LAHMY ET A L .
described by Tapiero et ~ l . RFLC3 , ~ being the most resistant ones. Once resistance was acquired, the cells were grown in a drug-free medium. The Gl,, (50 per cent inhibition of growth) for RFLCl, RFLC2 and RFCL3 were 20ng ml-’, 400ng ml-’, and 1300ng m1-l. They were cultivated routinely in RPMI 1640 (Flow) supplemented with 2 mM glutamine (Flow), 10 per cent of decomplemented foetal calf serum (Gibco), and antibiotics. All cell cultures were grown at 37°C in a CO, incubator. To maintain continuous exponential growth, cells were seeded at 0.1 x lo6 cells mland passaged every 2 or 3 days. All experiments were performed with cells in the exponential growth phase. In all experiments, the viability of the cells was measured by counting cells excluding 0.1 per cent trypan blue, and this was never below 95 per cent. The cell lines were tested regularly and found to be mycoplasma-free.
’
Exposure to Ho 33342
The chemicals used were all of analytical grade. Ho33342 (2’-(4-ethoxyphenyl)-5-(4-methyl1-piperazinyl)-2,5’-bi-l H-benzimidazole) from Aldrich was dissolved in phosphate-buffered saline (PBS) at l o p 3 mole per liter. The cell suspension (1 ml) was added to an aliquot of the stock solution. The final concentration of Ho33342 was 1 0 p in ~ the culture medium except where otherwise mentioned. Cells were incubated for 2 h at 37°C and then centrifuged (3 min, 100 y). They were rinsed three times with cold PBS (4°C). We then examined the cells in Sykes-Moore chambers without the upper lamella by image analysis. When necessary, verapamil from Sigma (10 PM, final concentration) was added to the cell suspensions 30min before the addition of Ho33342, and this was maintained during the incubation and rinsing. Fluorescence Spectra of Stained Nuclei
We recorded the fluorescence spectra of Ho33342 bound to DNA in solution, or accumulated in the nuclei of the sensitive and resistant cells using a microspectrofluorimeter built in our l a b ~ r a t o r y . ~The ~ . ~excitation ~ wavelength was 340 nm. Numerical Image Analysis
The number of sampled nuclei in each distribution was in the range of 300-350. The videomicro-
11
VIDEOMICROFLUORIMETRY OF MDR CELLS
Ruorimetry system has already been described.24 It consists of an inverted fluorescence microscope (Olympus, IMT2) equipped with an epi-illuminating system, a 40 x objective (Leitz), and a S.I.T. (Silicon Intensified Target) camera from Lhesa coupled to a TITN SAMBA 2002 image processor (see Figure 1). Depending on the excitation wavelength required, a mercury or xenon lamp can be used. For these experiments, a dichroic mirror selected the line 365 nm of a high-pressure mercury lamp (100 W) for fluorescence excitation. Quantitative measurements were possible, since a special data acquisition program was developed to avoid artefact^.'^ Precautions include the removal of the background, the correction of the non-linearity of the digitizer and the heterogeneity of the camera gain. pixel to pixel. This was made possible by using an homogeneous fluorescent reference sample for all experiments. Furthermore, this reference sample enabled us to compare one experiment with another.25Since Ho33342 is fluorescent only when bound to DNA, the fluorescence signal came from the nucleus. A protocol to record only the nuclear fluorescence was developed: image segmentation by thresholding, followed by a cell contour smoothing by opening and closing sequences. The use of parameters relative to the surface of the nuclei and
t
Figure 1.
Scheme of the videomicrofluorimetry apparatus.
to the fluorescence intensity makes rejection of subcellular debris, or cell clumps possible. RESULTS Staining Conditions
In every case the fluorescence properties of a probe must be studied and analyzed before quantification. Therefore a spectroscopic study of the fluorescence of stained nuclei has been performed. There was no apparent difference between the fluorescence spectra of the sensitive cells and those of the resistant ones. As shown in Figure2, there was no difference between the fluorescence spectrum of Ho33342 bound to DNA in solution or accumulated in the nuclei of resistant and sensitive cells. Thus it was assumed that the interactions between Ho33342 and its environment were similar whether Ho33342 was bound to DNA or accumulated in the cells. The conditions of quantitative measurements of Ho33342 fluorescence were next defined. In order to determine the concentrations to be used, two different concentrations of the probe 5 0 p ~and
12
S. LAHMY ET AL.
IF counts
i
400
500
600
h (nm)
Figure 2. Comparison of fluorescence spectra for sensitive (0) and resistant ( 0 )cells after staining with Ho33342 (10 PM) and Ho33342 bound to DNA in solution (.). Fluorescence spectra have been normalized to show the absence of shift. The spectrum of resistant cells has been multiplied by 4.
10 ,UM, were tested on the sensitive and the resistant cells. Figure 3 shows the distributions of fluorescence intensity for both concentrations of Ho33342 obtained for the sensitive cells (A,B) and the resistant cells (C,D). Comparison of the two doses on the sensitive cells showed that (1) after staining with the probe at 10 PM, a classical histogramz6 was observed with a peak corresponding to cells in G0/1 phase at 15000 counts and a peak corresponding to cells in G2 + M phase at 30000 counts; (2) after staining with the probe at 50 ,UM, unexpected results were obtained: the fluorescence intensities were in the same range of values as those obtained after staining at 10 ,UM although higher fluorescence intensities would have been expected. Furthermore, the distribution of fluorescence intensities was slightly shifted towards lower values. This could be explained by fluorescence quenching due to a high concentration locally. In the case of the resistant cells, no significant difference was observed between the two concentrations, but the resulting histograms were completely different from those for sensitive FLC: lower fluorescence intensities were obtained, as well as different cell distributions. However, the use of ethidium bromide has shown no difference in DNA content between the sensitive and the resistant cells.27Thus it was concluded that Ho33342 cannot
be used as an indicator for the cell cycle in the resistant cells. Therefore the choice was made to perform the experiments with 1 0 , to~ avoid ~ any perturbations or variations due to the concentration of the probe. Because staining time is a major variable, variation of nuclear fluorescence intensity as a function of incubation time was also studied. Both curves in Figure 4 show that after 2 h of incubation, staining reached a plateau and that there were no significant variations in the staining thereafter. Consequently, this period of incubation was chosen for the experiments on the sensitive and the resistant cells. These conditions allowed comparisons to be made between the fluorescence intensities of the cell populations because the staining conditions were equivalent. These curves also indicated that the fluorescence intensity (i.e. the amount of probe accumulated in the nucleus) was lower in the resistant cells than in the sensitive ones.
Accumulation of Ho33342 in Sensitive and Resistant Cells Having established the staining conditions, the accumulation of Ho33342 was studied in the different cell lines, all observed in their exponential growth phase: FLC sens, RFLC1, RFLC2 and
13
VIDEOMICROFLUORIMETRY OF M D R CELLS
t
t C
D
"'i
23.3
11.7
5.7
12000
IF
36000
12000
36000
Figure 3. Influence of drug concentrations in the staining. Cell distribution (%) as a function of fluorescence intensity 1a.u.) Sensitive--A: 10 pM, B: 50 L ~ MRFLC3-C: . 10 pM, D: 50 pM. I50000
T 5
100000
Ci
+.
.-*m
-
I
/
C
QJ
5
i
50000
I
1
0
0
1
2
3
4
5
6
hours Figure 4. Influence of incubation time for the staining of sensitive ( 0 )and resistant cells (+).
RFLC3 (Figure 5). The first peak of the distributions was used to compare the cell populations. For RFLC1, the distribution showed lower intensity values and a shift in the peak value of - 20 per cent compared to the peak obtained with
the sensitive cells. This shift increased for RFLC2 and reached -73 per cent for the peak of RFLC3. This represented a factor of 4 in the level of Ho accumulation between FLC sens and RFLC3. For clarity, the position of the peak of sensitive cell distribution is indicated by a dotted line in the four distributions. These results show that the accumulation of Ho33342 is inversely correlated to the resistance level. Figure 5 also shows that the values of fluorescence intensity for RFLCl were in the same range as the sensitive cells, but that there was a slight shift towards low values. The peak position in the distributions of RFLC2 and RFLC3 was completely shifted, and out of the range of the distribution of the sensitive cells. Nevertheless, some cells exhibited fluorescence intensities in the range of those of the sensitive cells. The number of these cells decreased with increase in cell resistance. Efeect of Verupumil
To test the relationship between low nuclear accumulation of Ho33342 and cellular resistance to ADR, we tested the effect of varapamil (VPL) on resistant cells (RFLC2, RFLC3). VPL is a calcium channel blocker and is often used to reverse multi-
14
S. LAHMY ET A L .
25.8
RFLC2
5.2 W
v
$?
A
12.8
RFLC3
7.7
2.6
6.9
0
12000
36000
IF
12000
36000
IF
) sensitive and resistant cells. Cell distributions (%) as a Figure 5 . Differences in accumulation of Ho33342 (10 p ~ for function of fluorescence intensity (a.u.). Dotted line indicates the peak of sensitive cells.
drug Figure 6 shows the distributions in the presence and absence of VPL for RFLC3. In the absence of VPL, the mean value of the population located under 12000 counts was 4741 with a standard deviation of 1642 counts. After addition of VPL, the mean value of the cell population under 12000 counts was 6670 with a standard deviation of 2192 counts. This shift represents an increase of 30 per cent of the fluorescence intensity: according to the Fisher-Snedecor test, the populations are different at 99 per cent.28 Similar results were found for RFLC2 (data not shown). However, in neither case did the intensities of fluorescence with VPL treatment reach the values obtained with the sensitive cells. This agrees with the findings of Morgan et aL2' Addition of VPL, at the same concentration to sensitive cells had no effect on the accumulation of Ho33342.
RFLC3
Deiection of Resistant Cells in a Mixture
To check the potential of this method, it was applied to the detection of resistant cells in a mixture of cells. Figure 5 shows that it would be difficult to detect t i e percentage of RFLCl in a
12000 36000 IF Figure 6. Effect of verapamil (10 PM) on the accumulation of Ho33342 ( 1 0 ~in~ RFLC3. )
VIDEOMICROFLUORIMETRY OF MDR CELLS
mixture of sensitive and RFLCl cells. For this reason a study was carried out on a mixture of RFLC3 and sensitive FLC. RFLC3 and sensitive cells were mixed at the same density in a ratio of 1 :2. Cells were incubated according to the procedure described in ‘Materials and methods’. The histogram of fluorescence intensities of the mixture is shown in Figure 7. Two groups of cells would be distinguished. The first had a value with a maximum at 4000 counts and represented about 35 per cent of the whole population. The second had its maximum at 16000 counts and represented 65 per cent of the cells. There was a factor 4 between the two peak values, which corresponds to that observed for the Ho33342 accumulation into the sensitive and the RFLC3. A similar experiment was also carried out with a mixture and the RFLC2 and sensitive cells (data not shown). Although it was possible to distinguish cells with a high resistance level, these measurements show that the method is not accurate enough for the cells with a low resistance level (namely RFLC1). DISCUSSION Different approaches have been used to detect the resistance of tumour cells to cytotoxic drugs. These include the presence of P-glycoprotein duc to MDR e x p r e s ~ i o n ; increased ~,~ glutathione transferase a ~ t i v i t y ; ’ ~ .and ~ ’ nuclear accumulation of fluorescent cytotoxic drugs.’,16 The disadvantage associated with the last method is the low quantum yield of the compounds, particularly ADR, when
12000
24000
IF
Figure 7. Distribution of fluorescence intensities for a mixture of sensitive and RFLC3 cells.
15
bound to DNA. Thus, for accurate measurements, high concentrations of the drugs have to be used in investigations on single living cells, but then it becomes difficult to distinguish toxic effects from cellular events. The advantages of the Ho33342 are that not only can low concentrations of the probe (10 j t instead ~ of 100 PM”) be used due to its high quantum yield of fluorescence, but also this vital stain does not affect living cells. However, in observations at 400 and 600 nm by flow cytometry, Morgan et al.” mentioned spectral shifts in the spectra of Ho33342 accumulated in the nuclei of sensitive and resistant small lung cells. We have therefore studied the fluorescence spectra of nuclear Ho33342 accumulated in sensitive and resistant FLC, before any comparison of fluorescence intensities by numerical image analysis were made. No differences were observed between these fluorescence spectra and the fluorescence spectrum of Ho33342 bound to DNA. This indicates that the molecular environment of the probe is similar in both cases. The results obtained indicate that the conditions described for the staining represent a convenient protocol. Comparison of the histograms of fluorescence intensities for RFLC1, RFLC2, and RFLC3 have shown (1) substantial differences between fluorescence intensities of the sensitive cells, RFLC2 (GIso = 400ng ml-’) and RFLC3 (GIso = 1300 ng ml- I); (2) only slight differences between sensitive cells and RFLCl (GI,, = 20 ng ml-’); (3) that Ho33342 cannot be used to monitor phases of the cell cycle in resistant cell lines, since accumulation of the probe depends on the resistance level. However, there was no difference in the DNA content between sensitive and resistant cells.27 The treatment of RFLC3 and RFLC2 by VPL, known as a resistance modulator, led to a higher accumulation of Ho33342. This indicates that the nuclear accumulation of this probe can be related to the resistance. We can thus use it as a marker for resistance, and the potentialities of this probe have been confirmed by the detection of resistant cells in a mixture of sensitive cells and RFLC3. The protocol should now be improved to determine the percentage of resistant cells in a mixture with better accuracy. Although Ho33342 appears to be a suitable probe to discriminate resistant cells with quite a high level of resistance (GIso 2 400 ng ml- l), the results emphasize the difficulties of discriminating the low level resistant cells (GI5o I 20 ng ml-I).
16
In future studies, Ho33342 will be used to follow the reversion of the resistance in RFLC2 and RFLC3, following addition of other modulators. The relationships between resistance and other cellular parameters such as pH, C a 2 + , Mg2+ and mitochondria1 rhodamine accumulation, will be studied using Ho33342 as the resistance indicator. Secondly, we will study the possibility of using Ho33342 for detection of cells with low resistance level. We therefore need to determine the lowest difference in nuclear fluorescence which will allow a reliable distinction between resistant and sensitive cells. This may be done by growing cells in a medium containing ADR (which induces resistance) and by simultaneously evaluating the amount of Ho33342 accumulated in the cells. ACKNOWLEDGEMENTS We are indebted to Professors E. Kohen and J. Hirschberg for their critical readings. We thank Dr Tapiero and Mrs Trincal for providing the cells and FrCdkric Bancel for helpful discussions. This work was supported by INSERM CRE NO899015 and by the ‘Ligue Francaise contre le cancer’, and the ‘P81e regional Languedoc Roussillon de Genie Biologique et Mkdical’. REFERENCES 1. Skovsgaard, T. and Nissen, N. I. (1982). Membrane transport of anthracyclines. Pharmac. Ther., 18, 293-31 1 . 2. Fojo, A., Shin-ichi, A., Gottesman, M. M. and Pastan, I. (1985). Reduced drug accumulation in multiple drugresistant human KB carcinoma cell lines. Cancer Res., 45, 3002-3007. 3. Lampidis, T. J., Bernal, S. D., Summerhayes, I. C. and Chen, L. B. (1982). Rhodamine 123 is selectively toxic and preferentially retained in carcinoma cells in uitro. Ann. N Y Acad. Sci., 99, 299-302. 4. Tapiero, H., Munck, J. N., Fourcade, A. and Lampidis, T. J. (1984). Cross resistance to rhodamine 123 in adriamycin and daunorubicin resistant Friend leukemia cell variants. Cancer Rex, 44,5544-5549. 5. Willingham, M. C., Richert, N. D., Cornwell, M. M., Tsuruo,T., Hamada, H., Gottesman, M. M. and Pastan, I. H. (1987). Immunocytochemical localization of PI70 at the plasma membrane of multidrug-resistant human cells. J . Histochem. Cytochem., 35, 1451-1456. 6. Juranka, P. F., Zastawny, R. L. and Ling, V. (1989). Pglycoprotein: multi-drug resistance and a superfamily of membrane associated transport proteins. FASEB J., 3, 2583-2592. 7. Roninson,I. B.,Patel,M.C.,Lee,I.,Noonan,K. E.,Chen,C. J., Choi, K., Chin, J. E., Kaplan, R. and Tsuruo, T. (1989). Molecular mechanism and diagnostics of multidrug resistance in human tumor cells. Cancer Cells, 7, 81-86.
S. LAHMY ET AL. 8.
9.
10.
11. 12.
13.
14. 15. 16.
17.
18.
19. 20.
21.
22.
23.
Bradley, G.,Juranka, P. F. and Ling, V. (1988). Mechanism of multidrug resistance. Biochem. Biophys. Acfa, 948, 87-128. Moscow,J.A., Fairchild,C. R., Madden,M. J., Ransom,D.T, Wieand, H. S., O’Brien, E. E., Poplack, D. G., Cossman, J., Myers, C. E. and Cowan, K. H. (1989). Expression of anionic glutathione-S-transferase and P-glycoprotein genes in human tissues and tumors. Cancer Res., 49, 1422- 1428. Tew, K. (1989). The involvement of glutathione S-tranferases in drug resistance. In: Anticancer Drugs (Tapiero, H., Robert, J. and Lampidis, T., eds) Colloque INSERM/ John Libbey Eurotext, pp. 103-112. Lampidis, T. J., Krishan, A., Planas, L. and Tapiero, H. (1986). Reversal of intrinsic resistance to adriamycin in normal cells by verapamil. Cancer Drug Deliu., 3,25 1-259. Yusa, K. and Tsuruo, T. (1989). Reversal mechanism of multidrug resistance by verapamil: direct binding of verapamil to P-glycoprotein on specific sites and transport of verapamil outward across the plasma membrane of K562/ ADM cells. Cancer Rex, 49, 5002-5006. Willingham, M. C., Cornwell, M. M., Cardarelli, C. O., Gottesman, M. M. and Pastan, I. (1986). Single cell analysis of daunomycin uptake and efflux in multidrug resistant and sensitive K B cells: effects of verapamil and other drugs. Cancer Res., 46, 5941-5946. Hedley, D. W., Hallahan, A. R. and Tripp, E. H. (1990). Flow cytometric measurement of glutathione content of human cancer biopsies. Br. J. Cancer, 61, 65-68. Herweijer, H., Van den Engh, G. and Nooter, K. (1989). A rapid and sensitive flow cytometric method for the detection of multidrug resistant cells. Cyfomefry,10, 463-468. Krishan, A. and Ganapathi, R. (1979). Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry. J. Histochem. Cytochem., 21, 1655-1656. Erba. E.. Ubezio. P.. Bronnini. M.. Ponti. M. and D’Incalci. M. (1988). DNA damage, cytotoxic effect and cell cycle perturbation of Hoechst 33342 on L1210 cells in vitro. Cytomefry,9, 1-6. Lalande, M. E., Ling, V. and Miller, R. G . (1981). Hoechst 33342 dye uptake as a probe of membrane permeability changes in mammalian cells. Proc. Nufl. Acad. Sci. U S A . , 78, 363-367. Kapunscinski, J. (1990). Interactions of nucleic acids with fluorescent dyes: spectral properties of condensed complexes. J. Histochem. Cytochem., 38, 1323- 1329. Ginot, L., Jeannesson, P., Angiboust, J. F., Jardillier, J. C. and Manfait, M. (1989). Interactions of adriamycin in sensitive and resistant leukemia cells. A comparative study by microspectrofluorometry. Studiu Biophysica, 104, 145- 153. Morgan, S. A,, Watson, J. V., Twentyman, P. R. and Smith P. J. (1989). Flowcytometric analysis of Hoechst 33342 uptake as an indicator of multidrug resistance in human lung cancer. Br. J. Cancer, 60,282-287. Salmon, J. M., Vigo, J. and Viallet, P. (1981). Microspectrofluorimttrie sur cellules vivantes isoltes: couplage d’un microspectrofluorimetre a un microordinateur. Innoo. Tech. B i d . Med., 2, 679-686. Lahmy, S., Salmon, J. M. and Viallet, P. (1987). Effect of ellipticine on M F O activity in single living 3T3 fibroblasts studied by microspectrofluorimetry. Anticancer Res., 7, 353-360.
17
VIDEOMICROFLUORIMETRY OF MDR CELLS
24. Lahmy, S., Salmon, J. M., Vigo, J. and Viallet, P. (1989). pHi and DNA content modifications after ADR treatment in 3T3 fibroblasts. A microfluorimetric approach. Anficancer Rex, 9, 929-936. 25. Vigo, I., Salmon, J. M., Lahmy, S. and Viallet, P. (1991). Videomicrofluorimetry: from qualitative to quantitative measurements. Anal. Cell. Path., 3, 145- 165. 26. Arndt-Jovin, D. J. and Jovin, T. M. (1977). Analysis and sorting of living cells according to deoxyribonucleic acid content. J . Histoehem. Cyfochern.,25, 585-589. 27. Lahmy, S., Salmon, J. M., Vigo, J. and Viallet, P. (1991). Accumulation of adriamycin in Friend leukemia cells: image analysis applied to resistance mechanisms. J . Cell. PharmacoL, 2, 18-23.
28. Lamotte, M. (1967). In: Initiation aux MPthodes Stafistique.< Masson and Co: Paris, pp 76-86. 29. Lee, F. Y., Sciandra, J. and Siemann, D. W. (1989). A study of the mechanism of resistance to adriamycin in v i m . Biochem. Pharmaco/., 38, 3697-3705.
Received in revisedforni 29 July 1991 Accepted 2 September 1991
