P-glycoprotein expression in Ehrlich ascites tumour cells after in vitro and in vivo selection with daunorubicin.

Fluctuation analysis experiments were performed to assess whether selection or induction determines expression of P-glycoprotein and resistance in the murine Ehrlich ascites tumour cell line (EHR2) after exposure to daunorubicin. Thirteen expanded populations of EHR2 cells were exposed to daunorubicin 7.5 x 10(-9) M or 10(-8) M for 2 weeks. Surviving clones were scored and propagated. Only clones exposed to daunorubicin 7.5 x 10(-9) M could be expanded for investigation. Drug resistance was assessed by the tetrazolium dye (MTT) cytotoxicity assay. Western blot was used for determination of P-glycoprotein. Compared with EHR2, the variant cells were 2.5- to 5.2-fold resistant to daunorubicin (mean 3.6-fold). P-glycoprotein was significantly increased in 11 of 25 clones (44%). Analysis of variance supported the hypothesis that spontaneous mutations conferred drug resistance in EHR2 cells exposed to daunorubicin 7.5 x 10(-9) M. At this level (5 log cell killing) of drug exposure, the mutation rate was estimated at 4.1 x 10(-6) per cell generation. In contrast, induction seemed to determine resistance in EHR2 cells in vitro exposed to daunorubicin 10(-8) M. The revertant EHR2/0.8/R was treated in vivo with daunorubicin for 24 h. After treatment, P-glycoprotein increased in EHR2/0.8/R (sevenfold) and the cell line developed resistance to daunorubicin (12-fold), suggesting that in EHR2/0.8/R the mdr1 gene was activated by induction. In conclusion, our study demonstrates that P-glycoprotein expression and daunorubicin resistance are primarily acquired by selection of spontaneously arising mutants. However, under certain conditions the mdr1 gene may be activated by induction.

Several reports indicate that transcription of the mdrl gene can be induced in cell lines. Thus. a varietv of environmental stresses and circumstances that affect cellular differentiation have been shown to induce m4drl gene expression (Chin et al. 1990a: Mickley et al. 1996. The anthracycline doxorubicin has been shown to induce expression of P-gp after only 24 h of contact with sensitive cells (Volm et al. 1991: Chevillard et al. 1992. The mdrl promoter has been shown to respond directly to treatment with anthracyclines or xinca alkaloids (Kohno et al. 1989). On the other hand. fluctuation analysis expenrments have indicated that activation of the mdrl gene in human sarcoma cells occurs as a result of a stochastic process consistent with mutational events .
The purpose of the present study was: (1) to use Luria-Delbruck fluctuation analysis to investigate whether daunorubicin (DNR) induced P-gp in sensitive Ehrlich ascites tumour cells (EHR2) by mutation or induction: (2) to investigate whether P-gp could be induced in EHR2 by 24 h treatment in vivo with DNR: (3) to investigate the expression of P-gp after 24-h treatment m X ivo with DNR in cells that are retreated after reversal of resistance.

Chemicals
Daunorubicin as hydrochloride w-as purchased from Farmitalia Carlo Erba. Milan. Italy. All other chemicals were of analytical grade.

Tumour cells
The murine Ehrlich ascites tumour cell line (EHR2) xas used in the experiments. The cell line was maintained as ascites tumours in mice by weekly transplantation of 1.5 x 10-cells per mouse and established as an in Xitro culture.
In vixvo induction of P-op in EHR' and EHR2/0.8/R cells respectively was studied by treatment of the mice wvith DNR 6 mg kg'. on the 6th day of transplantation. Investigation of these cells was performed after 24 h of contact w ith DNR.
The resistant subline EHR2IDNR+ was used for calibration of the Western blot assay and as a positixve control for immunocytochemical analysis (Nielsen et al. 1994).

Determination of drug resistance
Growth inhibition was evaluated by the tetrazolium dye colorimetric assay (MTT; 3-4-5-dimethyl-2-thiazolyl-2.5-diphenvl-2H-tetrazolium bromide: Tada et al. 1986 Determination of cellular proliferation Cellular proliferation was measured as described by Chen et al (1994). EHR2 cells and variant cells were seeded in 96 plates and incubated for 72 h at 370C wxith 5% carbon dioxide. Cell growth was estimated by the MYT colometric method (Tada et al. 1986 Fluctuation analysis experiments The Luria-Delbriick fluctuation analysis is a combined experimental and statistical method that allows one to distinguish  Catcheside (1951). Caulated according to Luria-DelbrOck (1943) as mnodified by Lea and Coulson (1949). cNot applicable, as the methodology requires at least one population with no surviving colonies. betmeen variant cells arising by rare spontaneous mutations and xariant cells arising through adaption to an environmental selection (Lunra and Delbruck. 1943). The analysis is based on the variation that is seen in the emergence of colonies from parallel cultures. If resistance is acquired by induction via druc exposure. the number of surviving colonies will be expected to haxe a Poisson distribution. with the variance equal to the mean. Thirteen tissue culture flasks (25 cm: Nunc A/S. Roskilde. Denmark) were seeded with EHR2 cells (2000 cells per flask) and allowed to grow (4.0 x lIW cells). Cells (2.6 x I0W) from each population were then seeded into separate 24-well plates with Nunc Tissue Culture Inserts (Anophore membranes: Nunc A/S) (two plates per population). After 48 h of incubation to ensure logarithmic growth. cells were treated with DNR 7.5 l-" si and 1 0-M respectivelv. The drug-containing medium w-as changed every day for 14 days and then replaced by drug-free medium.
These selection conditions were chosen based on preliminary expenments.
Surx-iving colonies were allowed to grow for 14 daxys and were then individually harvested and propagated in drug-free medium for further studies. In order to investigate the probabilitx of preexisting resistant clones in the original seeded population. populations of 2000 EHR2 cells were plated in separate 24-well plates and treated directly with DNR (DNR 7.5 10`N and 10sw: 26 plates per concentration) without expansion of the populations before drug exposure. In this experiment. no survixinc colonies were observed (data not shown).

Analysis of variance and mutation rates
The mutation rates were calculated according to the method of Catcheside ( 1951 )   where is the mutation rate per cell generation. r is the number of resistant colonies at time 1 and 2. N represents the initial number of cells adjusted for plating efficiency and gen is the number of generations. Furthermore. the PO method according to Luria and Delbruck (1943) as modified by Lea and Coulson (1949) was used: where PO represents the fraction of cultures with no mutants. i is the rate of mutations per cell generation and N, and No are. respectively. the final and the initial cell numbers. adjusted for the plating efficacy. As the last-mentioned method requires the presence of cultures with no variant cells. it could not be used to calculate the mutation rate in EHR2 cells exposed to DNR 7.5 10 m.

P-gp expression, Western blot analysis
Western blot analysis was used for semiquantification of P-gp (Nielsen et al. 1994). Briefly. cells were suspended in hypotonic buffer (sodium chloride 1O m-. magnesium chloride 6HW 0.15 nm-i. Tris 50 mn-i and phenylmethylsulphonylfluoride 2 mnst. pH 7.4). disrupted and centrifuged (4000g. 10 min). The supematants were centrifuted at 40 000 g for 60 min. The pellets were harxested. diluted and centrifuged (40 000g. 60 mmn). The membrane-enriched pellets were resuspended in buffer and loaded on the gels.  (1) Number of experiments given in parenthesis. two or three mice were used for every experiment.
The expression of P-gp was determined by reflectance photometrv. The relati've content of P-gp was calculated using a standard curve composed of membrane preparations with defined concentrations of P-gp (Nielsen et al. 1994).

P-gp expression, immunocytochemical analysis
For immunocytochemistry the APAAP technique described by Cordell et al (1984) was performed. Monolayers of cells on slides were air dried and fixed in ice-cold methanol-acetone (1:1).
Incubation was performned overnight with C2 19 at 40C. followed by incubation with rabbit antimouse IgG (Dako z259. Copenhagen. Denmark) for 30 min at 24°C and then by incubation with the (alkaline phosphatase anti-alkaline phosphatase) APAAP complex (Dako D65 1) for 30 min at 24°C. The last two steps were repeated for 10 min in order to amplify the signal. For washing Tris-buffered saline (pH 7.6) was used. Fast red was used as cromogen (Tablets. Kem-En-Tec. Copenhagen. Denmark).

Fluctuation analysis
The data presented in Table 1 show the number of surviving colonies in the plates (two plates per population) from each group studied by fluctuation analysis. In the group exposed to DNR 7.5 x 10-9 xi. the mean number of colonies sur iving per plate was 52. with a variance of 712. In the group exposed to DNR lO' M. the mean number of colonies surviving per plate was only 1.5. with a variance equal to the mean.
Determination of mutation rates Fluctuation analysis was also used to determine rates for DNR resistance in the EHR2 clones. According to the method of Catcheside ( 195 1 ). the mutation rate was estimated to be 4. 1 x I0o at DNR 7.5 x 1X9 M. whereas it decreased to 1.2 x l0-at a drug concentration of l0-mi.
Drug resistance, expression of P-gp A total of 25 clones were propagated from the group that was treated with DNR 7.5 x 10-9 M. At least one clone from each population was represented. All clones were stable. The resistance is shown in Table 2. Compared with EHR2 the degree of resistance varied from 2.5-to 5.2-fold (mean 3.6-fold). None of the resistant clones manifested any significant difference in generation time. Among the propagated clones 11 (44%) showed significantly increased expression of P-gp as compared with EHR2 (Table 2).
Nineteen clones were isolated in the group that was treated with DNR 108 M; however, none of these clones could be propagated for fturther studies.

Treabrment in vivo Sensitive Ehrlich ascites tumour cells
Sensitive EHR2 cells were treated in vivo with DNR; 6 mg kg-' for 24 h. The experiment was repeated five times using 2-3 mice every time. The expression of P-gp in teated cells was mean 15 ±8 (s.d.) arbitrary units. The expression of P-gp in EHR2 has previously been determined to be 12±4 units (Nielsen et al, 1994). In the present study, treatment with DNR for a short period of time in vivo did not induce P-gp in sensitive cells.
Revertant Ehrlich ascites tumour cells EHR2/).8/R was treated in vivo with DNR 6 mg kg-' for 24 h. The experiment was repeated six times using two or three mice every time. The expressions of P-gp are given in Table 3. In three instances the measurements were continued to day 15, in one instance P-gp expression was followed to day 240. The cell lines were passaged every seventh day in these periods. The expression of P-gp increased significantly in the treated tumours, showing the highest expression from days 15 to 45. Immunocytochemistry analysis was performed 1 day after treatment with DNR. This analysis showed a uniform weak immunoreactivity of cytoplasma and cell membrane of the EHR2/0.8/R cells, not different from EHR2 cells. None of the EHR2/0.8/R cells showed significantly increased expression of P-gp (data not shown).

DISCUSSION
Knowledge regarding the genetic and biochemical nature of drug resistance has been derived largely from cellular models developed by multistep, long-term drug exposure. Little is known about the initial genetic changes and relative frequencies of activation of various drug resistance mechanisms in tumour cell populations. The theory of genetic instability of tumours suggested by Goldie and Coldman (1979) proposes that drug-resistant cells emerge from the clonal expansion of spontaneously mutated cells, rather than from changes in cellular function induced by the drugs. This theory is based on the classic fluctuation analysis experiments performed by Luria and Delbrick (1943) in bacteria and by Law (1952) in mammalian tumour cells. There are fumdamental genetic differences between bacteria and somatic cells, specifically cancer cells. In addition, considerable statistical error is associated with the fluctuation analysis. The use and limitations of the analysis have previously been reviewed by Kendal and Frost (1988). They concluded that the method was appropriate in the field of somatic cell genetics. However, reliable results were only found regarding qualitative demonstration of consequences of variation.
After exposure to DNR 7.5 x 109 M the number of surviving EHR2 clones arising from 13 different populations showed a substantial fluctuation, as demonstrated in Table 1. The variance in the number of colonies was 14-fold greater than the mean, indicating that, under these experimental conditions (5 log cell killing), clonal survival of the cells was a consequence of the selection of spontaneous mutations in the expanded cell populations. The number of resistant colonies that arise in fluctuation analysis is dependent on the rate of mutation, the generation time of the variant cells and the time of appearance of the variant cells.
Moreover, the clones that are scored must arise during the expansion of the parallel cultures (Kendal and Frost, 1988). If a resistant cell was present in the initial seeding of 2000 EHR2 cells, at least 2048 progeny would have been present after 11 generations of growth. The highest number of surviving colonies among our expanded populations was 87, indicating that the event responsible for the cell survival occurred after a minimum of five generations of population expansion. Furthermore, the pre-existence of resistant variants in the initial cell population was unlikely, as none of the cell clones survived when the parental EHR2 cells were treated with DNR without prior expansion.
Tlhe literature is generally unclear on the precise events regulating the appearance of P-gp-mediated resistance. Both induction and selection have been described, with no compelling evidence prented to support definitively one or other mechanism as overriding in all cellularhumour models. Prior studies have been performed in many different cells lines, using different drugs, exposures and analytical approaches. It is possible that the various conclusions apparent in the literature could reflect these different experimental conditions. On the other hand, Chen et al (1994) used fluctuation analysis to investigate the resistance mechanisms in human sarcoma (MES-SA) cells exposed to the anthracycline doxorubicin. In accordance with our result, these authors demonstrated that spontaneous mutations conferred resistance to doxorubicin. These authors also used the Luria and Delbriick fluctuation analysis to investigate resistance mechanisms in MES-SA cells exposed to paclitaxel (Dumontet et al, 1996), etoposide ) and a combination of cyclosporin PSC 833 and doxorubicin (Beketic-Oreskovic, 1995). In all instances except one, analysis of variance supported the hypothesis of spontaneous mutations conferring resistance. In MES-SA cells exposed to a very high dose of etoposide (5 piM), analysis of variance showed the variance equal to the mean . This latter finding was compatible with our finding in EHR2 cells exposed to DNR 10 M, suggesting that at highly stringent conditions of selection (6 log cell killing) the resistance could be a result of adaption.
In the present study, the increase in P-gp was very modest; further, P-gp increased in only 44% of the surviving clones. In contrast, Chen et al (1994) in MES-SA cells exposed to doxorubicin observed increased mRNA mdrl in all clones tested . The expression of P-gp in these cells was, however, modest, in accordance with our results (3-9% compared with control, MDR cells). In addition, Dumontet et al (1996) demonstrated increased rndrl mRNA in only 4 of 9 clones of MES-SA clones exposed to paxlitaxel. Besides overexpression of the mdrl gene, which is the predominant mechanism of resistance to DNR. cells can manifest resistance to this agent by decreased expression and/or altered activity of topoisomerase II. Other reported mechanisms of resistance include alteration in cellular glutathione level. expression of multidrug resistance-associated protein (MRP) or expression of lung resistance protein (LRP) (Nielsen et al. 1996).
Compared with EHR2. the degree of resistance of the surviving clones was modest (2.5 to 5.2-fold). This finding is in agreement with the finding of Jaffrezou et al (1994). who used etoposide as selecting arent. In the previously mentioned studies the degrees of resistance were. however. found to be significantly higher . Betetic-Oreskovic et al. 1995. Dumontet et al. 1996.
The rate of mutation conferring resistance to DNR w-as 4.1 x 10per cell reneration. The statistical errors associated with variation rate measurements are considerable (Kendal and Frost. 1988). On the other hand. the mutation rate was in the range of spontaneous point mutations described for mammalian cells (10--10per cell generation) (Borst. 1991) and comparable with that previously described in human sarcoma (MES-SA) cells . Dumontet et al. 1996: but lower than that described for some gene amplifications. ranging from 1 to 7 x 10-' per cell generation (Crawford et al. 1983. Tlsty et al. 1989).
In the present study. P-p could be induced in EHR2/0.8/R by 24 h treatment with DNR in vivo. but not in sensitive EHR2 cells.
The inducibility of the mdrl gene and P-gp by treatment with cytostatics has been investigated bv several authors (Kohno et al. 1989: Chin et al. 1990b: Chaudharv and Roninson. 1993: Fardel et al. 1997. In contrast to our finding. P-gp could be induced in sensitive cells. Kohno et al (1989) found that the mdrl promoter w-as activated by vincristine. anthracyclines and colchicine. Chin et al (1990b) demonstrated substantially increased mdrl mRNA in rodent cell lines after acute cvtostatic treatment in vitro.
Chaudhary and Roninson (1993) reported significantly increased mdrl gene expression and increased resistance (2to 3-fold) in several human tumour cell lines. which were exposed transiently (12 h to 5 days) to chemotherapeutic drugs in vitro. Furthermore. in L 12 10 tumour cells treated in vivo with doxorubicin 0.5 mg kg-' for 10 min to 96 h. Volm et al (1991) measured the expression of P-gp by immunohistochemistrv. They reported 85-90%c P-gp-positive cells 24 h after treatment. These authors. however. found only a slirht increment in resistance. Recently. Chevillard et al (1992) reported increased P-gp expression to be induced in sensitive human lung adenocarcinoma cells (A549) after 24 h treatment with doxorubicin. The emerguence of P-gp was follow ed by approximately three-fold resistance to doxorubicin. P-gp has been found to be dose dependently induced in a human pleural mesothelioma xenograft (PXF 1118) (Licht et al. 1991). In this cell line. P-gp was induced in absence of proliferation. thus favouring the proposal that increased expression of P-gp is associated with phenotypic cell change rather than resulting from selection of a pre-existing drugy-resistant subpopulation.
In general the degree of resistance induced in sensitive cells after transient treatment with cytostatic was low (2 to 3-fold) (Licht et al. 1991. Volm et al. 1991: Chevillard et al. 1992). In contrast. the EHR2/0.8/R cell line developed sig'nificant resistance to DNR ( 11.9-fold). Previously. the expression of P-gp in revertant cell lines has only been investigated by Gekeler et al (1988). In an actinomycin A-selected human leukaemia cell line maintained 1 month without selection pressure. mdrl gene transcription was inducible after 72 h of retreatment. In this cell line the degree of resistance was. how7ever. not investigated.
In the present study. immunocvtochemistrv showed a uniform Aweak immunoreactivitv of the treated EHR20.8/R cells. not significantly different from EHR2. The expression of P-gp was probably below the detection himit of the immunocvtochemistrv assay. Thus. this finding suggested that P-gp was induced in a high fraction of the EHR2/0.8/R cells. In spite of cessation of treatment P-gp increased further after transplantation of EHR2/0.8/R. reaching, the highest level from days 15-45. This high expression of P-gp was comparable with the expression of P-gp in EHR2 cells. which had been exposed to multiple doses of DNR (Nielsen et al. 1994). This finding could be in accordance with either a direct activation of the mdrl promoter as described by Kohno et al (1989) or an indirect activation of the promoter by interaction with a regulatory protein (Gant et al. 1992). sugaesting that the activation of the mdrl gene occurred by induction.
In conclusion. our study demonstrates that P-gp expression and DNR resistance are primarilv acquired by selection of spontaneously arising mutants. However. under certain conditions the mdrl gene could be activated by induction. ABBREVIATIONS DNR. daunorubicin: MDR. multidrugy resistance: P-gp. P-glycoprotein