Determination of DNA topoisomerase II in newly diagnosed childhood acute lymphoblastic leukemia by immunocytochemistry and RT-PCR

CANCER LETTERS Cancer

Letters

84 (1994) 141-147

Determination of DNA topoisomerase II in newly diagnosed childhood acute lymphoblastic leukemia by immunocytochemistry and RT-PCR G. Stammlera,

A. Sauerbreyb,

M. Volm*”

“German Cancer Research Center, 0511, Im Neuenheimer Feld 280. P.O. Bou 101949. 69009 Heidelberg, Germany bChildrens University Hospital of Jena, Jena, Germanj

Received

10 May 1994; accepted

16

June 1994

Abstract

The expression of DNA-topoisomerase II was analysed at the protein level in newly diagnosed cases of acute lymphoblastic leukemia (ALL) in children. Blast cells obtained from 81 children with untreated ALL were determined by means of immunocytochemistry. Of the ALL, 49 (60%) were positive for topoisomerase II and 32 (40%))negative. No significant correlation was found between the expression of topoisomerase II and the relapse rate or relapse-free intervals. These results were substantiated by determining the topoisomerase II mRNA expression in a collective of 21 patients by semiquantitative PCR. The PCR-assay and immunocytochemistry corresponded in 13 of 21 cases (62%). Kevwords;

DNA topoisomerase II; Acute lymphoblastic

1. Introduction Among leukemias, childhood acute lymphoblastic leukemia (ALL) is one of the most sensitive to cytotoxic chemotherapy. ALL patients treated with combinations of various antineoplastic drugs often go into remission. The response rate of ALL is approximately 95%, but 25% of the patients relapse and rezidives become drug resistant. Resistance to chemotherapeutic agents often results from decreased intracellular drug accumulation caused by in* Corresponding author. 0304-3835/94/$07.00 0 1994 Elsevier Science Ireland SSDI 0304-3835(94)03476-Y

leukemia; Immunocytochemistry;

RT-PCR

creased drug efflux, ascribed to an overexpression of P-glycoprotein or detoxification by glutathioneS-transferase x [l-3]. Overexpression of proteins like O6 alkylguanin-DNA-alkyltransferases, thymidylate synthase or others may also play a role in resistance to chemotherapeutica. A number of antineoplastic drugs such as intercalating anthracycline, acridine, ellipticine, anthracenediones, [4-71 and non-intercalating epipodophyllotoxine [8,9] affect topoisomerase II. This enzyme regulates the topology of DNA and is involved in replication, transcription, and chromosome segregation [lo]. The drugs stabilize the transient cleavable complex

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of the enzyme, which becomes covalently bound to the DNA during the breakage-reunion reaction [8]. The formation of this stable ternary complex (DNA-enzyme-drug) leads to DNA damage and ultimately in cell death [ 111. Thus, cells with decreased topoisomerase II activity could result in minor response to antineoplastic drugs when compared with cells exhibiting high topoisomerase II levels. Qualitative or quantitative alteration in topoisomerase II activity in multidrug resistant cells was reported by several authors [ 12- 161. Because the intercalating drug daunorubicin was included in chemotherapy of the examined ALL, we suggest that the activity of topoisomerase II may play a role in the development of resistance. Therefore, in the present study we determined the topoisomerase II content from acute lymphoblastic leukemia cells from 81 children, prior to chemotherapy, by immunocytochemistry and compared the results with the clinical outcome after treatment with cytotoxic agents. To substantiate these results, we developed a sensitive semi-quantitative PCR assay and examined the topoisomerase II mRNA expression in 21 of the 81 patients.

I-aparaginase followed by a consolidation therapy with cyclophosphamide, cytarabine, 6-mercaptopurine and intrathecal methotrexate. Patients received intermediate-dose intravenously methotrexate (0.5 or 1 g/m2) and/or cranial irradiation. Maintenance therapy was performed with oral 6-mercaptopurine (daily) and methotrexate (weekly) up to 2 years after starting therapy.

2. Materials and methods

2.4. Immunocytochemistry The biotin-streptavidin-peroxidase-method was used for detection of topoisomerase II. Cell suspensions were centrifuged in a cytospin 2 (Shandon) resulting in a cell monolayer. The cells were air dried, fixed in ice-cold aceton for 10 min and stored at -20°C. The slides were preincubated with normal goat serum to block unspecific binding sites. The polyclonal topo II antibody (topoGen; Columbus, OH) (1: 100) was applied for 16 h at 4°C in a moist chamber. This antibody recognizes the 170kDa form (topoisomerase II cu). After washing, the cells were incubated with biotinylated goat antirabbit second antibody (Dianova, Hamburg, Germany) and 5% normal human serum for 15 min. After subsequent washes in phosphate-buffered saline, the streptavidin-biotinylated-peroxidase complex (Amersham, Braunschweig, Germany) was added and the peroxidase activity was visualized by 3-amino-9-ethylcarbazole. Counterstaining was performed with Mayers hematoxylin. To suppress endogenous peroxidase and biotin activity, the samples were preincubated with 0.3% Hz02 and

2.1. Patients Eighty-one children with acute lymphoblastic leukemias (ALL) from the Childrens’ University Hospital of Jena were studied. Diagnosis of leukemia were performed by standard cytological and histochemical examination of bone marrow and blood smears according to the French-American-British (FAB) classification and by immunological investigation of the blast cells using indirect immunofluorescence.’ Fourteen leukemias were classified as pre B-ALL, 37 as C-ALL and 25 as T-ALL. Five patients were not available for immunophenotyping. Sixty-two of the children were older and 19 younger than 10 years. Thirty-eight were male, 43 female. 2.2. Therapy All patients received therapy according to a cooperative modified BFM-protocol [ 17,181. This treatment protocol consists of an induction therapy with prednisone, vincristine, daunorubicin, and

2.3. Cells Leukemic cells were obtained from bone marrow or peripheral blood. Cell samples were collected in heparinised flasks and mononuclear cells were isolated by Ficoll Hypaque density gradient centrifugation. After washing twice in culture medium (RPM1 1640), the cells were cryopreserved in liquid nitrogen with 10% DMSO and 5% FCS using a programmed freezer. All cell samples contained at least 80% blast cells, determined by May Gri.inwald Giemsa staining. Sarcoma 180 (S180) cells were grown intraperitoneally (i.p.) in ascites form in female NMRI mice. The development of a doxorubicin-resistant subline was described previously [ 191.

G. Stammler et al. /Cancer

unlabelled streptavidin. First, negative controls were carried out by omitting the primary antibody and, second, by substituting the primary antibody with an irrelevant, isotype-matched antibody. The immunocytochemical staining was graded either as negative, weakly positive, moderately positive, or strongly positive according to a score which we have previously validated in a series of animal as well as human cell lines and human solid tumors. 2.5. RNA isolation and Northern blot analysis Leukemic cells were rapidly thawed in a 37°C water bath and centrifuged at 1000 x g for 5 min. A roral of lo7 cells were lysed in 2 ml RNA-Clean (AGS, Heidelberg, Germany) and RNA was extracted following the instructions of the manufacturer. Finally, the RNA pellet was resolved in DEPC-HIO. Only RNA samples with clear rRNA bands were used. A IO-pg quantity of total RNA was transferred to Hybond N (Amersham) and Northern hybridization was carried out following the recommendations of the manufacturer. 2.4. Reverse trancription (RT)-PCR assay A l-p1 quantity of random hexamers (50 ng/pl) was added to 2 pg RNA and the RNA was reverse transcribed following the instructions of the Superscript Preamplification System from Life Technologies (Karlsruhe, Germany). The reverse transcription mixture (20 ~1) was diluted with the same volume of HZ0 and 2 ~1 (50 ng cDNA) was added to 48 ~1 PCR-mix. The PCR-mix contained 5 ~1 10 x reaction buffer (100 mM Tris-HCl, 15 mM MgCl,, 500 mM KCl; pH 8.3), 2 ~1 dNTP-mix (each dNTP 2.5 mM), 1 ~1 each primer (10 PM), 0.25 ~1 5 U/l Taq-Polymerase (Boehringer, Mannheim, Germany) and 38.75 ~1 H,O. A 2-min denaturation step at 94°C was followed by 20 (µglobulin as internal standard) or 25 (topoisomerase II) cycles of primer annealing (55°C 40 s), primer extension (72°C 60 s) and denaturation (94°C 40 s). Because it was necessary to estimate the amount of PCR products in a linear range and the linear region of amplification of &-microglobulin and topoisomerase II did not coincide the standard and topoisomerase II DNA were amplified separately. Numerous negative controls were included in each

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143

set of PCR reactions to detect any possible contamination. The controls were constructed by using the cDNA synthesis mixture as described above without the addition of reverse transcriptase or contained water instead of cDNA. PCR-products were separated in 1.8% agarose gels and transferred to Hybond N (Amersham) following the instructions of the manufacturer. The membranes were prehybridized for 1 h at 65°C in prehybridization buffer (1 .O M NaCl, 50 mM Tris-HCl (pH 7.5), 10% dextransulfate, 1% SDS, 100 fig/ml denatured salmon sperm DNA). 5’-end labeled 40mer oligonucleotides were added and hybridized at 65°C overnight. After washing four times briefly at room temperature and two times for 15 min at 65°C in 2 x SSC, 0.1% SDS the membranes were autoradiographed at -80°C on Kodak X-OMAT AR film. The Southern hybridization of the PCR-products with oligonucleotides ensured that the target sequence was amplified and increased the sensitivity compared with ethidiumbromide-staining several-fold. Therefore, we were able to use a lower cycle number so that we did not run the risk of getting into the plateau phase. A Hirschmann elscript 400 densitometer was used for scanning the autoradiographs. Only samples with nearly the same amounts of internal standard amplification product were included in the examination because slight or very dense bands on the autoradiograms tend to be out of the linear range of the densitometer. 2.7. Primer and oligonucleotides To minimize the influence of variations in RNA quality, the primers (20mers) were chosen so that only small fragments of topoisomerase II (289 bp) and &-microglobulin (261 bp) were amplified. The sequences of the primers (20mers) were for topoisomerase II a! (specific for mouse and human) 5 ‘TGG TCA GAA GAG CAT ATG AT-3 ’ and 5 ‘CTC ACA ATC TGA TCA GCT AC-3 ‘, for j32microglobulin (human) 5 ‘-AAG ATG AGT ATG CCT GCC GT-3 ’ and 5 ‘-TCA AAC ATG GAG ACA GCA CT-3’ [20], for &-microglobulin (mouse) 5 ‘-ATG GGA AGC CGA ACA TAC TG3 ’ and 5 ‘-CCA GTA GAC GGT CTT GGG CT3 ’ . The oligonucleotides (40mers) used for hybridization were 5 ‘-TAC CAG TTT CAT CCA ACT TGT CCT TCA AAT ACA TGT CCA C-3’ for

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topoisomerase II Q, 5 ‘GCA AGC AGA ATT TGA ATT CAC TCA ATC CAA ATG CGG CAT C-3’ for &-microglobulin (human), and 5’-TAG AAA GAC CAG TTC TTG CTG AAG GAC ATA TCT GAC ATC T-3 ’ for &-microglobulin (mouse). 2.8. Statistical analysis We used Fisher’s exact test to examine whether there is a correlation between clinical data and topoisomerase II expression. Life table analyses according to Kaplan and Meier [21] were performed for relapse-free intervals. For the statistical analysis the immunocytochemical specimens were estimated only as negative or positive. Cell samples were graded as negative when there was complete absence of staining, whereas each positive reaction was classified as positive.

Fig. 1. Immunocytochemical treated acute lymphoblastic positive cells.

staining of topoisomerase II in unleukemia (ALL) of children. Arrow:

3. Results The aim of the study was to analyse the role of topoisomerase II as a prognostic factor in untreated newly diagnosed childhood acute lymphoblastic leukemia. Therefore, we evaluated the expression of topoisomerase II at the protein level by immunocytochemistry and at the mRNA-level by RT-PCR. Eighty-one leukemias were examined by immunocytochemistry. A typical expression pattern of topoisomerase II in leukemia cells is shown in Fig. 1. Of the 81 patients 49 (60%) were classified as topoisomerase II-positive and 32 (40%) as negative. Fourteen (44%) cases of topoisomerase II-negative leukemias relapsed, whereas 19 (39%) positively stained cases experienced relapses. Fig. 2 shows the relapse-free interval curve of the patients. The topoisomerase II expression was not correlated with the relapse-free interval. In order to confirm these results, the topoisomerase II mRNA expression was investigated in a group of 21 patients by semiquantitative RT-PCR. The suitability of the PCR-assay was first tested with doxorubicin sensitive and resistant S180 ascites cells. Preliminary studies showed, with the help of immunocytochemistry, that topoisomerase II level was decreased in doxorubicin-resistant versus doxorubicin-sensitive S180 cells [ 121. The data of the PCR-assay corresponded with these tindings: sensitive S180 cells yielded in higher

topoisomerase III& microglobulin ratio than doxorubicin-resistant S 180 cells. The results were also in accordance with Northern blot analysis (Fig. 3). In leukemic cells the topoisomerase II 289 bp fragment was amplified in all samples except one. Fig. 4 shows a representative autoradiogram. The topoisomerase III&-microglobulin ratio was calculated and the samples were classified as either

Fig. 2. Relapse-free intervals (Kaplan-Meier estimates) grouped of topoisomerase II.

of patients with ALL according to the expression

G. Sfunimler

S top0

R

II

cl (11./ Cuncer Lett. 84 (1994)

S

R

Table I Results of immunocytochemistry

m-

standard

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(ICC) and RT-PCR

PCR-

PCR+

Total

ICCICC+

5 5

3 8

8 13

Total

10

II

21

Samples classified as topoisomerase II-negative by PCR are described as PCR- and -positive as PCR+. Samples classified as topoisomerase II-negative or -positive by immunocytochemistry are described as ICC- and ICC+.

A

B

Fig. 3. Expression of topoisomerase II mRNA in doxorubicin sensitive (S) and resistant (R) S180 cells estimated by Northern hybridization (A) and RT-PCR (B). &Actin was used as internal standard for Northern hybridization and /3z-microglobulin for RT-PCR.

cells with increased and low topoisomerase II mRNA content by estimation of the median. The results of the PCR-assay corresponded in 13 (62%) cases with the immunocytochemical analysis and differed in 8 cases (Table I). The clinical analysis with these 21 samples revealed no significant correlation between topoisomerase II content and relapse rate by either immunocytochemistry or RT-PCR (Table 2). 4. Discussion In the present study we have investigated the correlations between topoisomerase II protein and

a

mRNA expression and response to chemotherapy in childhood acute lymphoblastic leukemia by immunocytochemistry and RT-PCR. Decreased topoisomerase II activity in drug resistant cell lines was often described in the past. Kim et al. found that the topoisomerase II mRNA expression was significantly correlated with clinical response in solid human tumors [22]. Several studies showed that topoisomerase II levels are low in chronic lymphocytic leukemic cells (CLL) [23-261. The authors suggested that the resistance of CLL to doxorubicin is due to the extremely low levels of topoisomerase II in these cells. In a recent study of topoisomerase II CYand /3 expression in adult acute myelogenous leukemia, it was shown that both isozymes had no significant effect on drug sensitivity [27]. In general, reports about the activity of topoisomerase II in ALL are rare. Gekeler et al. found no significant correlation between topoisomerase II mRNA expression and responsiveness to chemotherapy in ALL blast cells [23]. In agreement with these studies we observed neither a significant correlation between topoisomerase II expression and the clinical outcome. These results are not unexpected, because

Table 2 Classification topoisomerase cytochemistry

Fig. 4. Representative autoradiogram the corresponding µglobulin described RT-PCR-assay.

of topoisomerase II (a) and (b) in ALL obtained by the

No relapse Relapse

of relapsed and not II-positive and -negative (ICC)

relapsed patients as by PCR and immuno-

PCR-

PCR+

ICC-

ICC+

4 6

7 4

5 6

6 4

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et al. /Cancer

there are other drugs than topoisomerase II inhibitors involved in the BFM-protocol. One part of our study was the comparison of two methods, immunocytochemistry and RT-PCR. In comparison with RT-PCR, immunocytochemistry offers the advantage of being a simple and fast method. Additionally, immunocytochemistry allows detection of the cell-to-cell heterogeneity of topoisomerase II expression. Because we could detect topoisomerase II in 20 of 2 1 samples by PCR but only in 49 of 81 by immunocytochemistry we think that the described PCR assay is the more sensitive method. The data obtained by immunocytochemistry and RT-PCR corresponded in 62% and neither had a significant correlation with the relapse rate. However, we do not have a explanation of why both methods did not confirm completely. This may be found in the variability and different sensitivity of both methods or in mRNA and protein level diversity. Such disparities in the estimation of the protein and mRNA level have been frequently described for the mdrl gene [28,29]. A comparison of PCR and immunocytochemistry with the estimation of the topoisomerase II activity (unknotting, relaxation and catenation/decatenation activity) could be helpful in determining which method is superior to the other. Thereby, posttranslational modifications like ribosylation and phosphorylation would also be considered. References

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