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Significance of fluorescent histogram of daunorubicin(DNR) in

¡¡¡¡diagnosis of drug resistance to leukemic cells

He Ming£­sheng, CHEN Yan, WU Yu£­dan, et al

¡¡¡¡¡¾Abstract¡¿Objective£ºTo develop a fluorescent histogram of intracellular daunorubicin(DNR) as a diagnostic method for drug resistance in acute myeloid leukemia(AML).Methods£º15 patients with clinically diagnosed drugª² resistant AML and 5 patients with previously untreated AML were studied. The intensity of fluorescence of intracellular DNR and the fluorescent histogram were determined by flow cytometer(FCM).According to a degree of the intensity, both drug£­resistant and£­sensitive zones were marked off. Left shift of main peak(LSMP) with the fluoresent histogram may be diagnosed as drug resistance, and right shift of main peak(RSMP) as drug sensitivity. R/S stood for drug resistant index(DRI);R for the number of drugª² resistant cells, and S for drug£­sensitive cells. DRI¡Ý 2 was drug£­resistant or DRI £¼2 was drug-sensitive. Fluorescent index(FI) was the product of mean fluorescent intensity (MFI) multiplied by DRI. FI¡Ý 200 meant drug-resistant, while FI£¼ 200 meant drug-sensitive.Results£ºAmong 5 patients with previously untreated AML, 4 patients were diagnosed as drug-sensitive for RSMP, DRI="0.23¡«" 1.13,FI="50¡«" 160,and one patient was diagnosed as primary drug-resistant for LSMP,DRI="3.60," FI="367." In 15 patients with clinically drugª² resistant AML,12 patients were diagnosed as classical resistance for LSMP,DRI="2.59¡«" 17.72,and FI="265¡«" 1041,and 3 patients as regrowth drug resistance for RSMP,DRI="0.39¡«" 0.67,and FI="57¡«" 83.Conclusion£ºClinically, it was a rapid, convenient and visual way, which by FCM mapping the fluorescent histogram of intracellular DNR, in combination with DRI, FI, a clinically drug-resistant manifestation and a cutª² off point for diagnosis of the fluorescent intensity of drug-resistant cells, we may in good time find and value a type and a degree of drug resistance to AML. Moreover ,it may provide a basis for working out an individually therapeutic scheme and a method for an observation in drug-resistant reversal treatment as well.

¡¡¡¡Key words£ºDaunorubicin(DNR); Fluorescent histogram; Drug resistance; Flow cytometer (FCM); Leukemia

¡¡¡¡Drug resistance in leukemia was a leading cause underlying chemotherapeutic failure. Its degree and its type were close associated with the chemotherapeutic scheme for each patient. Still, an urgent problem to solve in clinic is how to find and evaluate the resistance quickly, conveniently and timely. Daunorubicin (DNR) produces red fluorescence with laser excitation at 488 nm wavelength. Utilizing it, drug resistance was detected through measuring DNR fluorescent intensity in the cells. It was only in a few minutes that flow cytometer(FCM) could analyse it for thousands of cells and draw into a fluorescent histogram that represented the marks of drug resistance to leukemic cells. Hence, we tried to investigate the diagonstic significance for the fluorescent histogram in classical and in regrowth drug resistance in leukemia.

¡¡¡¡1¡¡MATERIALS AND METHODS

¡¡¡¡1.1¡¡Patients

¡¡¡¡15 patients with acute myelogenous leukemia(AML),who manifested drug resistance clinically according to a clinically drug£­resistant standard^¡²1¡³,were eligible for this study.9 males,6 females, age 17¡« 52 years old. 5 patients with previously untreated AML were investigated as control.3 males,2 females, age 32¡« 48 years old. All of them were diagnosed with a clinical, cell morphology and cell chemistry method in our hospital. Mononuclear cells were obtained with a standard way, i.e. Ficollª² Hypaque density gradient separation, in which the number of leukemic cells were more than 90£¥.

¡¡¡¡1.2 ¡¡Reagents

¡¡¡¡DNR(Farmitalia Carlo Erba,Italy) and RPMI 1640(Gibco£­BRL,USA).

¡¡¡¡1.3¡¡Assay of DNR fluorescent intensity in leukemic cells

¡¡¡¡Leukemic cells were washed twice with RPMI 1640 which contained 10£¥ calf serum. The concentration of cells was adjusted to 1¡Á 10⁶ /ml.1 ¦Ìg/ml DNR was added and incubated for 1 hour at 37¡æ ,5£¥ CO2.Then dissociated DNR was washed twice with RPMI 1640 at 4¡æ. 1 ml RPMI 1640 at 4¡æ was added, the fluorescent intensity of cells was detected by FCM ( Becton Dickinson, USA ). 488nm wavelength was used. FCM was first adjusted with fluorescent balls , and CV value was lower than 3£¥.10 000 cells were assayed for each sample. Sample without DNR was regarded as control. The detected results were drawn into the histogram with software CELLQuest according to fluorescent intensity that represented intracellular DNR concentration. The ordinate was cell number and the abscissa was logarithmic fluorescent intensity.

¡¡¡¡1.4¡¡Data analysis

¡¡¡¡According to other article^¡²2¡³,the cut-off point of DNR fluorescent intensity in drug£­resistant cells was 100. Passing through this point, fluorescent intensity was divided into two zones, namely, £¼100 was the zone of drug resistance, and ¡Ý100 was the zone of drug sensitivity. The left shift of main peak(LSMP) in DNR fluorescent histogram meant that it located in the drug£­resistant zone. The previously untreated patient was diagnosed as primary drug resistance. If the patient manifested drug resistance clinically, he was diagnosed as classical drug resistance. The right shift of main peak £¨RSMP£©meant that it located in the drug£­sensitive zone. If the patient manifested drug resistance clinically, he was diagnosed as regrowth drug resistance. R stood for cell number of drug resistance;S stood for cell number of drug sensitivity. The drug resistant index(DRI)="R/S." If it¡Ý 2,it meant drug resistant, and£¼2 meant drug sensitive. Fluorescent index (FI)="mean" fluorescent intensity(MFI)¡Á DRI. FI£¼200 indicated sensitivity, and¡Ý200 indicated resistance; the larger the value of them, the higher the degree of drug resistance.

¡¡¡¡2¡¡RESULTS

¡¡¡¡The DNR fluorescent histogram. 4 cases of previously untreated AML displayed RSMP in the fluorescent histogrom, in which the main peak were high£­narrowed(Fig.1), DRI=0.23¡«1.13,and FI=50¡«160.These patients were diagnosed as drug sensitive. One case of previously untreated AML displayed LSMP, in which the main peak was high£­narrowed, too (Fig.2),DRI=3.60,and FI=367. The patient was diagnosed as primary drug resistant.15 cases of AML showed drug resistance clinically, in which 12 cases displayed LSMP(Fig.3), including in 3 cases represented a subpeak near the main peak(Fig.4), DRI=2.59¡«17.72, and FI=265¡«1041.These patients were diagnosed as classical drug resistance. 3 cases of AML who showed drug resistant clinically displyed RSMP, in which the shape of the main peak was between high£­narrowed and low£­broadened peaks, DRI=0.39¡« 0.67,and FI=57¡« 83. They were diagnosed as regrowth drug resistance(Fig.5).

DNA fluorescent intensity(log) ¡¡¡¡Fig1	DNA fluorescent intensity(log) ¡¡¡¡Fig2	DNA fluorescent intensity(log) ¡¡¡¡Fig3
¡¡¡¡¡¡¡¡¡¡ DNA fluorescent intensity(log) ¡¡¡¡Fig4 DNA fluorescent intensity(log) ¡¡¡¡Fig5

¡¡¡¡Fig.1¡¡The newly diagnosed patient with RSMP.

¡¡¡¡Main peak was highª² narrowed.He was diagnosed as drug sensitivity.

¡¡¡¡Fig.2¡¡The newly diagnosed patient with LSMP.

¡¡¡¡Main peak was highª² narrowed,too.He was diagnosed as primary drug resistance.

¡¡¡¡Fig.3¡¡The patient showed drug resistance clinically.

¡¡¡¡There was obvious LSMP.He was diagnosed as classical drug resistance.

¡¡¡¡Fig.4¡¡The patient showed drug resistance clinically.

¡¡¡¡Apart from main peak,in low fluorescent zone,there was another peak,and it suggested that there were two different cell subpopulations which had different degrees of drug resistance. Mean fluorescent intensity was 121.04.The main peak located in the centre.Its shape was low£­broaded,DRI=2.60,and FI=314.He was diagnosed as classical drug resistance.

¡¡¡¡Fig.5¡¡The patient showed drug resistance clinically.

¡¡¡¡There was obvious RSMP.It suggested that he was still sensitive to the drugs.He was diagnosed as regrowth drug resistance.

¡¡¡¡Abbreviation

¡¡¡¡RSMP:right shift of main peak;LSMP:left shift of main peak;DRI:drug resistance index:FI:fluorescence index;DRZ:drugresistant zone;DSZ:drug sensitive zone

¡¡¡¡3¡¡DISCUSSION

¡¡¡¡The mechanism of drug resistance to leukemic cells was very complex,one of which, chemotherapeutic agents may be pumped out of these cells. In some confirmed experiments, in vitro measurements of DNR concentration functionally disclosed in vivo drug resistance to cells. FCM could determine the DNR fluorescent intensity which represented the variations of the DNR concentration in cells. As some experiments had been demonstrated, there was an inverse proportion of the intensity to the concentration^{¡²2,3,4,5¡³}.

¡¡¡¡At present, MFI often means drug resistant to a cell population, and it depends on two factors, i.e. the fluorescent intensity and the cell number which generate fluorescence. DRI could account for the degree of drug resistance. The larger the value of it, the higher the degree. Meanwhile, referred to Webb's method^¡²6¡³£¬FI accounted for the degree of drug resistance. For example, firstly, a patient in this study was diagnosed as drugª² sensitive for MFI=121.04 but secondly, he was diagnosed as drug resistant after a combination of DRI=2.60 and FI=314 was considered. Con£­sequently, the second diagnosis was consistant with his manifestation of clinical drug resistance.

¡¡¡¡Leukemic cells in vivo belong to a heterogenous population with different degrees of drug resistance. From the fluorescent histogram, definite number of resistant cells were presented in all the patients, previously untreated or clinically drug£­resistant, whose number will make a characteristics or a phenotype of drug resistance. Besides the main peak, one or two subpeaks were sometimes seen in the drug resistant zone. This explained the presence or development of resistant subpopulations, and also explained the heterogenicity of drug£­resistant cells.Because they could survive the chemotherapeutic agents, there was some prognostic significance in observing the variations of their number underlying relapse and drug resistance in leukemia. Additionally, the alteration of their degree and their type of drug resistance, to some extent, would ensue from the growing of cell colonies in leukemia.

¡¡¡¡From what we had observed, there were two main peaks in the histogram£ºone was a high£­narrowed peak that meant the resistant or sensitive cells were concentrated in a certain degree; the other was a lowª² broadened peak that meant the cells were dispersive. The former, including drug£­resistant andsensitive cells which centered on a certain phase, may have a good effect on agents. The latter, which cells had different degree of drug resistance, may have a poor effect on agents. Furthermore, the more left£­shifting of the main peak, the higher the degree of drug resistance. As for secondary drug resistance, the main peak, which its shape varied from high£­narrowed to low£­broadened and its position varied from right to left, may suggest that the drugª² sensitive cells have changed into the drug£­resistant cells.

¡¡¡¡FCM can in a few minutes determine the DNR fluorescent parameters of ten thonsands cells and draw the fluorescent histogram and it takes four hours or so to reach the analytic conclusion. So, it provides a rapid, convenient and visual basis for designing individually therapeutic regimens and provides a method for a clinical observation in reversing drugresistant treatment as well.

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¡¡¡¡REFERENCES

¡¡¡¡1¡¡Hiddemann W and Buchner T. Treatment strategies in acute myeloid leukemia(AML)¡²J¡³. Blut,1990,60£º163¡«171.

¡¡¡¡2¡¡He Yi£­xin, Yang Shao£­xian, Zhang Shu£­jing, et al. Identification and isolation of multidrug tolerance(MDT) cells¡²J¡³. Chin J Hematol,1992, 13£º173¡«175.

¡¡¡¡3¡¡Kokenberg E, Sonneveld P, Delwel R,et al. In vivo uptake of daunorubicin by acute myeloid leukemia(AML) cells measured by flow cytometry¡²J¡³. Leukemia,1988,2£º511¡« 517.

¡¡¡¡4¡¡Xu D, Knaust E, Pisa P, et al. Levels of mdr1 and mrp mRNA in leukeamic cell populations from patients with acute myelocytic leukemia are heterogenous and inversely correlated to cellular daunorubicin accumulation¡²J¡³. Br J Haematol,1996,92£º847¡« 854.

¡¡¡¡5¡¡Guerci A, Merlin JL, Missoum N, et al. Predictive value for treatment outcome in acute myeloid leukemia of cellular daunorubicin accumulation and P£­glycoprotein expression simultaneously determined by flowª² cytometry¡²J¡³. Blood,1995,85£º2147¡«2153.

¡¡¡¡6¡¡Webb M, Raphael CL, Asbahr H, et al. The detection of rhodamine 123 efflux at low levels of drug resistance¡²J¡³. Br J Haematol,1996,93£º650¡« 655.

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