• acute myeloid leukaemia;
  • cytosine arabinoside;
  • cytidine deaminase;
  • drug resistance


  1. Top of page
  2. Abstract
  6. Acknowledgements
  7. References

Gene transfer of the cytidine deaminase (CDD) cDNA has recently been shown to induce cellular resistance to cytarabine (AraC) in vitro. To investigate the role for CDD in acute myeloid leukaemia (AML) we analysed the CDD activity and CDD gene structure in blast material from well-defined patients with untreated and AraC refractory (RF) AML. Median CDD activity in previously untreated AML was significantly lower than in RF-AML blasts (P = 0.015) and was significantly lower in patients with complete remission than with blast persistence following induction chemotherapy (P = 0.043). Structural investigation of the CDD gene by Southern analyses and RT-PCR showed no detectable aberrations. Sequence analysis of the CDD cDNA from nine RF-AML patients showed inconsistent aberrations in three patients. Semiquantitative assessment of CDD mRNA expression revealed a significant correlation with CDD activity. In conclusion, concordant with another recent study our data suggest a correlation of pretherapeutic CDD activity with induction treatment response. Besides the previously described prognostic impact of mdr1 expression, this result could be useful for the development of risk-adapted AML treatment strategies and warrants further studies of CDD activity in well-defined cohorts of AML patients and of the mechanisms involved in the regulation of CDD activity.

Cytostatic induction chemotherapy of adult acute myeloid leukaemia (AML) with daunorubicin or idarubicin plus cytarabine (AraC) results in complete remission rates of 60–80%. In certain subgroups of patients long-term disease-free survival (DFS) rates of around 40–60% are achieved with myeloablative regimens followed by allogeneic haemopoietic progenitor cell transplantation (alloPCT) or with intensive post-remission regimens including high-dose AraC (HD-AraC) ( Mayer et al, 1994 ). In unselected cohorts of AML patients, however, DFS rates are in the range of 20–25%. Thus, cytostatic drug resistance towards anthracyclines and AraC remains the major obstacle to long-term DFS in the majority of AML patients.

Further improvement of AML therapy is conceivable with the development of risk-adapted treatment strategies based on pretherapeutic individual or tumour biological determinants. Prognostic factors which have been previously established are age at diagnosis and karyotypic aberrations, and may possibly include antecedent haematological disorders, LDH levels, and autonomous growth of leukaemic cells in vitro ( Mrózek et al, 1997 ; Mayer et al, 1994 ; Büchner et al, 1993 ; Hunter et al, 1993 ). Theoretically, among the most sensitive predictors for treatment response should be those associated with mechanisms of cellular resistance towards anthracyclines and AraC. In fact, although not yet firmly established, there is accumulating recent evidence that the expression of an mdr1 phenotype in leukaemic blasts correlates with anthracycline resistance and is an adverse prognostic factor for reaching a complete remission and long-term DFS ( Schröder et al, 1996a ; Campos et al, 1992 ; Nuessler et al, 1997 ; Del Poeta et al, 1996 ; van den Heuvel-Eibrink et al, 1997 ; Kasimir-Bauer et al, 1998 ). In contrast, although several putative in vitro mechanisms of cellular AraC resistance have been identified during the last two decades, controversy exists as to their potential clinical impact. Among others, these include an inactivation of deoxycytidine kinase (DCK; EC and an increase of cytidine deaminase (CDD; EC activity ( Capizzi et al, 1991 ).

Circumstantial evidence for a possible correlation between CDD activity and AraC resistance has been suggested by previous in vitro and in vivo studies ( Yusa et al, 1992 ; Honma et al, 1991 ; Riva et al, 1992 ; Kreis et al, 1977 ; Steuart & Burke, 1971; Colly et al, 1987 ; Jahns-Streubel et al, 1997 ; Tattersall et al, 1974 ). A causal relationship between these variables in vitro, however, has only recently been established by us and other groups ( Schröder et al, 1996b ; Neff & Blau, 1996; Momparler et al, 1996 ; Flasshove et al, 1997 ). In addition, we showed that recombinant proteins corresponding to two natural variants of the CDD cDNA which differ by a single amino acid are associated with significantly different deamination rates of AraC in vitro ( Kirch et al, 1998 ). As these data may suggest a structure–function relationship that could be relevant to clinical AraC resistance, we performed a structural analysis of the CDD gene in blasts from patients with newly diagnosed and recurrent AML. Considering the inconsistent data obtained from previous in vivo studies investigating the potential correlation of CDD or DCK activities with clinical AraC resistance, we also assessed the CDD and DCK enzymatic activities in well-defined subsets of AML patients with either sensitive, persistent or recurrent disease.


  1. Top of page
  2. Abstract
  6. Acknowledgements
  7. References

Patients and treatment

From 1993 to 1996, bone marrow (BM) or peripheral blood (PB) samples were obtained from consecutive, uniformly treated patients suffering from de novo AML. In 35 patients blast samples were obtained at the time of initial diagnosis, and in 36 patients at the time of first or subsequent relapse ( Table I). Diagnosis, classification and remission status of AML were assessed according to standard criteria ( Cheson et al, 1990 ). Patients with antecedent haematological disorders, including myelodysplasia or known exposure to carcinogens, were excluded from the analysis.

Table 1. Table I. Patients' characteristics.Thumbnail image of
  • a

    * Favourable/normal/unfavourable/not done ( Mrózek et al, 1997 ).† uc: unclassified.

  • Eligible patients received first-line protocol treatment consisting of one or two induction courses of idarubicin at a dose of 12 mg/m2 administered on days 1–3 as a 30 min infusion, and of AraC at a dose of 100 mg/m2 as intravenous bolus injection on day 2, followed by continuous AraC infusion at a dose of 200 mg/m2 on days 2–6. Post-remission therapy within this protocol consisted of one treatment cycle identical to the induction therapy given above, and of a second consolidation course with AraC at a dose of 3 g/m2 every 12 h, days 1–4, and cyclophosphamide at 1000 mg/m2 on days 1 and 3; for patients > 50 years of age respective doses in the second consolidation course were adjusted to 1 g/m2 AraC and 750 mg/m2 cyclophosphamide. Treatment at disease relapse consisted of AraC at a dose of 1–3 g/m2 plus either daunorubicin, mitoxantrone and/or etoposide. Granulocyte- or granulocyte-macrophage colony-stimulating factors (G-/GM-CSF) were not routinely employed before, during or after induction or consolidation therapy. Refractory AML in the context of this analysis was classified as recurrent disease from previous complete remission following the aforementioned treatment including intermediate- to high-dose AraC (1.0–3.0 g/m2) consolidation, or as blast persistence (> 25% BM blasts) following first-line induction therapy.

    Cell preparation

    Patients' samples eligible for structural and functional analysis had to have a percentage of leukaemic blasts of geqslant R: gt-or-equal, slanted 80–90% as determined microscopically and/or by FACS analysis of surface markers. For DNA and/or RNA isolation, samples were lysed in 4 m guanidine isothiocyanate immediately after aspiration and processed further for nucleic acid isolation as previously described ( Schröder et al, 1996b ; Chomczynski & Sacchi, 1987). In order to allow for RNA preparation from small volume blood samples (25–30 μl) a commercial RNA preparation kit (QuickPrep Micro mRNA Purification Kit, Pharmacia Biotech, Freiburg, Germany) was used for isolation of polyadenylated mRNA from some patients. RNA obtained from either procedure was resuspended in 50 μl of DEPC-treated water containing 10 U of RNAsin (Serva, Heidelberg, Germany) and immediately processed for further use or stored at −70°C. For enzyme analyses, samples were collected in heparinized tubes and mononuclear cells were isolated within 2–3 h after sample acquisition by Ficoll-Hypaque density gradient centrifugation (density < 1.077 g/ml; Pharmacia, Uppsala, Sweden). Cells were washed three times with cold phosphate-buffered saline, collected by centrifugation, suspended in 200 μl of 5 m m Tris-HCl, pH 7.4, and lysed by four cycles of freeze-thawing. After centrifugation at 12 000 g for 15 min, the Tris-HCl (pH 7.4) concentration of the supernatant was adjusted to 50 m m. Supernatant was immediately processed or stored at −70°C for up to 1 week until enzyme assay. Serial determinations of enzyme activities from freshly taken cell samples could be performed in three patients during the course of their disease and were not possible in the other patients because of technical constraints, e.g. insufficient sample size (see below) or protein instability in frozen cell extracts after long-term storage. With only a few exceptions, the volume/cell content from eligible blast samples was insufficient as to allow for simultaneous structural and enzymatic analyses; thus, the corresponding results of this study represent different groups of patients.

    Southern blot analyses

    High molecular weight DNA was isolated as described above. Southern blot analyses were performed with XbaI- or Eco RI-digested genomic DNA of patients for whom at least 2 μg DNA were available. Analysis of the methylation status of the CDD structural gene was done by restriction enzyme digests with MspI and HpaI. Restriction fragments were separated on 0.8% agarose gels and transferred to nylon membranes (Hybond N+; Amersham Buchler, Braunschweig, Germany) in 0.4 m NaOH. Hybridizations were done according to standard conditions using a 32P-dCTP-labelled (Multiprime kit; Amersham Buchler) 749 bp CDD cDNA fragment comprising the CDD open reading frame (ORF) and part of the 3′-untranslated region ( Schröder et al, 1996b ). Membranes were washed at 65°C using standard protocols and exposed for 2–5 d at −70°C to Kodak XAR-2 films.

    The CDD cDNA probe was obtained by RT-PCR from RNA of a healthy human donor as previously described and cloned into the EcoRI-site of plasmid pCDNA3 (Invitrogen, Leek, The Netherlands) ( Schröder et al, 1996b ). Identity of the CDD cDNA was verified by DNA sequencing using an A.L.F. DNA sequencing apparatus (Pharmacia) using vector-specific primers.

    Reverse transcription and polymerase chain reaction (RT-PCR)

    Reverse transcription, PCR conditions and oligonucleotide primers for CDD-specific PCR reactions were described previously ( Schröder et al, 1996b ). Oligonucleotide primers were custom synthesized by Pharmacia Biotech (Freiburg, Germany).

    Integrity of RNA and adequate cDNA synthesis was confirmed by using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) specific primers (Clontech, ITC Biotechnology GmbH, Heidelberg, Germany) as previously reported ( Schröder et al, 1996b ). Negative PCR controls consisted of reactions containing water instead of cDNA, and RNA without reverse transcription prior to PCR. Positive controls consisted of plasmid containing CDD-cDNA as described ( Schröder et al, 1996b ). PCR amplification products were analysed on 1–1.5% agarose gels using HindIII-digested lambda-DNA as a size marker.

    CDD mRNA expression was semiquantitatively assessed as follows: 2 μg of total mRNA were reverse transcribed in 50 μl reaction volume. PCR reactions were performed in 30 μl final volume as previously described containing 5 μl of RT reaction mix and 10 pmoles and 20 pmoles each of the GAPDH- and CDD-specific primers, respectively ( Schröder et al, 1996b ). PCR consisted of 29 amplification cycles at 94°C for 1 min, 62°C for 1 min, 72°C for 2 min, and a final extension step for 5 min at 72°C. Amplification products were separated on a 1% agarose gel, visualized by ethidium bromide staining, and photographed on Polaroid films. Optical densities (OD) of CDD- and GAPDH-specific bands were determined densitometrically. The signal intensity of the CDD amplification product was calculated as the ratio of ODs for CDD and GAPDH.

    DNA sequencing

    CDD-specific RT-PCR amplified cDNA products from nine patients with recurrent high-dose AraC resistant AML were cloned into the pGEM-7Zf(+) plasmid or pUC18 vector and transformed into E. coli strain DH5α. White colonies were isolated and analysed after DNA preparation and restriction digest for presence of the 749 bp CDD cDNA. A median of three clones was isolated per patient and subjected to sequence analysis of the entire CDD cDNA open reading frame using an A.L.F. DNA sequencing apparatus (Pharmacia) using vector-specific primers and the following internal CDD-specific primer 5′-TGGCTGTTACAGGCAGTGGGC-3′. In addition, the 5′-portion of the CDD ORF from 15 additional patients was custom sequenced at Eurogentec, Seraing, Belgium, using RT-PCR products and the internal CDD-specific primer 5′-GTTCAGCACAGATGCCCAG-3′.

    Enzyme assays

    CDD- and DCK-specific enzyme assays and control reactions were performed as previously described using 50 μg of cellular protein ( Schröder et al, 1996b ). Enzymatic analyses were performed in triplicate reactions except in two patients for whom material for only one reaction was available. Enzyme activities are expressed as nmol substrate conversion per hour at 37°C and given per mg protein present in the cell extract.

    Statistical analysis

    The Wilcoxon, Mann and Whitney U-test was used for comparison of enzyme activities between subgroups of AML patients. The Spearman two-tailed correlation analysis was applied for analysis of the relationship of CDD enzyme activities with remission duration, and the relationship of CDD enzyme activities with signal intensities of CDD RT-PCR amplification products.


    1. Top of page
    2. Abstract
    4. RESULTS
    6. Acknowledgements
    7. References

    Southern blot analysis

    Southern blot analyses were performed with XbaI- and EcoRI-digested genomic DNA from blast samples of 39 AML patients comprising 11 previously untreated patients and 28 patients with refractory (RF-AML), i.e. recurrent or primary resistant disease. Patterns and intensities of restriction fragments observed in all patients resembled those obtained with DNA from healthy human donors and placenta DNA indicating that no amplifications or other gross genomic aberrations had occurred (data not shown). For both the XbaI- and EcoRI-digestion, one restriction-fragment-length-polymorphism (RFLP) was detected within the CDD gene (Fig 1). A correlation of RFLP-allele distribution with disease status or treatment response was not observed. Analysis of the methylation status of the CDD structural gene using MspI- and HpaI-digested DNA was performed in eight untreated patients and nine patients with recurrent AML, and did not reveal differences between both groups of patients (data not shown).


    Figure 1. Fig 1. Representative Southern blot analysis showing XbaI-specific (left panel) and EcoRI-specific (right panel) RFLPs of the human CDD gene using genomic DNA from human placenta (HP) and patients' samples. Arrows on the left indicate fragment sizes. Hybridization was performed using a 749 bp CDD cDNA fragment (see Patients and Methods).

    Download figure to PowerPoint

    RT-PCR analyses

    RNA samples were available from 17 untreated patients and 27 RF-AML patients for CDD-specific RT-PCR. Visualization of amplification products on agarose gels (Fig 2) did not reveal alterations in transcript sizes when compared with amplification products obtained from RNA of a healthy donor and from a CDD-specific control plasmid.


    Figure 2. ); arrows indicate the 560 bp fragment of the lamda/HindIII size marker (M, lane 1) and the CDD-specific 755 bp amplification product.

    Download figure to PowerPoint

    CDD mRNA expression was semiquantitatively assessed by RT-PCR in 13 patients for whom CDD activities covering a wide range of enzyme activities were available (Fig 3). There was a significant correlation of CDD signal intensity with enzyme activity (r = 0.66; P = 0.014). No significant correlation was observed between CDD mRNA PCR intensity and FAB type (P = 0.086).


    Figure 3. AML patients. Relative ODs are given as the ratio of CDD- and GAPDH-specific signal intensities (correlation coefficient, r = 0.66; P = 0.014).

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    Nucleotide sequence analysis of CDD cDNA clones

    As no gross structural aberrations of the CDD gene were detected with the above-mentioned analyses, the CDD coding region was amplified by RT-PCR from blast samples of nine patients with recurrent HD-AraC-resistant AML and cloned into a plasmid vector. A median of three clones (range 1–4 clones) per patient were isolated and subjected to DNA sequence analysis of the CDD ORF. As previously reported, we noted a genetic polymorphism at nucleotide position 79 of the CDD ORF resulting in a non-conservative amino acid deviation (Gln[LEFT RIGHT ARROW]Lys) at codon 27 ( Kirch et al, 1998 ), corresponding to the CDD sequences described by Kühn et al (1993 ) (Gln) and Laliberte & Momparler (1994) (Lys), respectively. In a first preliminary approach to evaluate the distribution of Gln and Lys alleles among the AML patients we amplified the CDD cDNA coding region by RT-PCR and sequenced the 5′ portion of the ORF from another five patients with recurrent AML and from 10 untreated AML patients. Together with the sequence data from cloned CDD cDNAs of the nine patients mentioned above, no striking difference in allelotypes between patients with responsive/untreated and refractory AML was detected.

    Besides the polymorphism at codon 27, deviations from the published nucleotide sequences were observed in four of the nine patients ( 2 Table II). One patient (no. 37) showed a silent base pair mutation at nucleotide position 435. Nucleotide alterations leading to amino acid replacements were detected in two patients, affecting codons 55, 59, 97 and 135 in two of four clones in patient 3, and codon 115 in two of four clones from patient 29. In the fourth patient (no. 22) an internal deletion was detected in one of three clones analysed, spanning nucleotide positions 154–323. This deletion results in an ORF frameshift at codon 52 and a premature translational stop at nucleotide position 416 of the original CDD ORF sequence ( Table II). Among the four patients showing nucleotide aberrations, a protein sample for determination of CDD activity was available only for patient 22 and showed a CDD activity of 640 nmol × 10−3/h × mg protein.

    Table 2. Table II. Structural aberrations of the CDD cDNA coding region.Thumbnail image of

    Enzyme assays

    Table 3. Table III. CDD and DCK enzyme activities in patients with previously untreated and recurrent AML and in relation to induction treatment response.Thumbnail image of
  • a

    a P = 0.015; b P = 0.043; c P = 0.002; d P > 0.05. * Enzyme activities are given as nmol × 10−3/h × mg protein for CDD and as nmol/h × mg protein for DCK.† Included six early deaths.

  • Of the 36 patients analysed for CDD activity, five patients were ineligible for parallel DCK assays due to insufficient amounts of blast material. Thus, enzyme assays for determination of DCK activity were performed in 20 and 11 patients with untreated and recurrent AML, respectively, and revealed no significant difference (P > 0.05; 3 Table III). Likewise, no significant difference in median DCK activities was detected among the subgroups of patients with CR, blast persistence and recurrent disease ( 3 Table III). No significant correlation of CDD or DCK activity with karyotype, FAB classification or age among subgroups of patients was observed.


    1. Top of page
    2. Abstract
    4. RESULTS
    6. Acknowledgements
    7. References

    Among the various mechanisms involved in cellular AraC resistance, several lines of evidence have indicated a role for DCK deficiency and/or CDD increase in its pathogenesis in vitro ( Meyers & Kreis, 1978; Bhalla et al, 1984 ; Richel et al, 1990 ; Kees et al, 1989 ; Rustum & Preisler, 1979; Owens et al, 1992 ; Yusa et al, 1992 ; Honma et al, 1991 ; Riva et al, 1992 ; Kreis et al, 1977 ). Corresponding clinical studies, however, yielded inconsistent results ( Steuart & Burke, 1971; Colly et al, 1987 ; Jahn-Streubel et al, 1997 ; Tattersall et al, 1974 ; Mejer & Nygaard, 1978; Smyth et al, 1976 ), which at least in part may be explained by differences in treatment schedules, AraC doses and response criteria, variations in enzyme assays, and the inclusion in several studies of patients' samples with blast percentages as low as 30–50% possibly confounding enzyme activities with those of normal haemopoietic cells ( Ho, 1973). Further evaluation of the putative role of these enzymes in the pathogenesis of AraC resistance has only recently become possible with the molecular cloning of their corresponding cDNAs ( Chottiner et al, 1991 ; Kühn et al, 1993 ; Bertling et al, 1993 ; Laliberte & Momparler, 1994). In fact, gene transfer studies clearly established a causal relationship between enzyme activity and cellular sensitivity (DCK) or resistance (CDD) to AraC in vitro ( Manome et al, 1996 ; Schröder et al, 1996b ; Neff et al, 1996 ; Momparler et al, 1996 ; Flasshove et al, 1997 ). Furthermore, structural analyses showed inactivating mutations of the DCK gene as a cause of DCK deficiency in vitro and revealed polymorphism at codon 27 of the CDD cDNA to correlate with significantly different deamination rates of AraC in vitro ( Owens et al, 1992 ; Stegmann et al, 1995 ; Kirch et al, 1998 ). So far, a molecular analysis of these genes in primary human AML blasts has been reported only for the DCK gene. In a study of AraC-resistant AML patients we previously showed that DCK cDNA mutations occur only rarely in vivo and therefore may not constitute a major mechanism of clinical AraC resistance ( Flasshove et al, 1994 ).

    In the present study we first assessed DCK enzyme activities in patients with untreated, responsive and refractory disease and found no significant differences between these groups of patients. Although this result contrasts with data from some anecdotal studies, it is in accordance with another recent investigation ( Jahns-Streubel et al, 1997 ). Thus, as both our structural and functional analyses failed to indicate a role of DCK for AraC resistance in vivo, we next addressed the putative role of the CDD in cellular AraC resistance.

    Possibly influenced by the methodological reasons cited above, previous studies of CDD activity in primary human AML blasts had yielded inconsistent results, either showing a significant correlation of treatment failure with (a) increased CDD activity ( Steuart & Burke, 1971), (b) high CDD or low DCK activity ( Colly et al, 1987 ), (c) a non-significant trend for increased CDD activity ( Tattersall et al, 1974 ), or (d) no such relationship ( Mejer & Nygaard, 1978; Smyth et al, 1976 ). In the present investigation of uniformly treated AML patients we observed a significantly higher median CDD activity in recurrent AML than in previously untreated patients and also found a significant correlation of median CDD activity with induction treatment response. Interestingly, these results are concordant to those of another recent study of uniformly treated, well-defined AML patients describing a significant correlation of CDD activity with early blast cell clearance ( Jahns-Streubel et al, 1997 ). Whereas the latter investigation also suggested a correlation of CDD activity with remission duration, this relationship, however, failed statistical significance in our analysis.

    To elucidate the mechanism(s) associated with increased CDD activity, we performed Southern analyses in patients with refractory AML and found no indication for gene amplification or other genomic aberrations. Likewise, RT-PCR analyses covering the CDD ORF did not reveal aberrations in transcript sizes between patients with untreated, responsive or refractory disease. We next investigated the possible occurrence of nucleotide alterations within the CDD cDNA gene in AraC refractory patients. This analysis was based on our previous finding of the functional relevance of a single nucleotide exchange at codon 27 of the CDD cDNA gene ( Kirch et al, 1998 ). In addition, Stegmann et al (1995 ) had shown that AraC exposure of cells in vitro may lead to random mutations of the DCK gene with subsequent induction of AraC-resistant cell clones. As this, in analogy, might suggest the possible induction and selection for activating mutations within the CDD gene, we sequenced the CDD ORF in nine AML patients with recurrent HD-AraC-resistant AML. The internal deletion detected in one of three clones from one of these patients had no corresponding detectable amplification product in the RT-PCR analysis, thus questioning its functional relevance. Alignment of the unique amino acid mutations observed in part of the clones from the other two patients with the well-characterized E. coli CDD suggests that these mutations are located outside of functionally important domains ( Betts et al, 1994 ). Hence, whereas we have not yet determined the enzymatic activities of these mutated clones and no detailed structure–function analysis of human CDD is presently available, the functional impact of these mutations may be questionable.

    Since structural aberrations of the CDD cDNA gene did not seem to represent a major cause of the observed differences of CDD activities between AraC-sensitive and AraC-resistant patients, we performed a semiquantitative assessment of CDD mRNA expression and found a significant correlation of signal intensities for PCR amplification products with CDD enzyme activities. This result is in accordance with recent preliminary data from another group ( Jahns-Streubel et al, 1997 ) and suggests that CDD activity is at least partly regulated at the transcriptional level. Because in vitro incubation of AML cells with the hypomethylating agent 5′-aza-2′-deoxycytidine was reported to lead to increased CDD expression ( Laliberte & Momparler, 1994; Momparler & Laliberte, 1990), we also analysed the methylation status of the CDD structural gene but found no differences in the patterns of restriction fragments between the different groups of patients. Since the regulatory region of the CDD gene has not been cloned, the latter experiments, however, do not yet allow conclusions on the potential impact of promoter methylation on the regulation of CDD mRNA expression.

    In conclusion, consistent with data from several in vitro analyses and another recent clinical investigation of uniformly treated, well-defined AML patients the current study sheds further light on the possible relationship of CDD activity with AraC resistance in vivo. As to the establishment of risk-adapted strategies for AML therapy it may suggest further evaluation of the correlation of pretherapeutic CDD activity with early treatment response. Considering the high failure rate and the significant toxicity associated with HD-AraC consolidation therapy, it may as well warrant an evaluation of the relationship of pretherapeutic CDD activity with treatment outcome following HD-AraC containing post-remission therapy. To our knowledge, it represents the first structural analysis of the CDD gene in AML patients and indicates that CDD activity in vivo is correlated with transcriptional regulation rather than with CDD gene aberrations. Further insight into the regulation of CDD activity and its possible relationship to AraC resistance may be achieved by determination of codon 27 allelotypes in significantly larger number of patients and with cloning and analysis of the regulatory regions of the CDD gene.


    1. Top of page
    2. Abstract
    4. RESULTS
    6. Acknowledgements
    7. References

    This work was supported by Deutsche Krebshilfe/Dr Mildred Scheel-Stiftung, Bonn, Germany (grant W 18/94 Schü1). The authors thank Dr B. Opalka for advice in the performance of multiplex PCR for the semiquantitative analyses of CDD mRNA expression, Ms Jutta Tins for excellent technical assistance, Dr Kasimir-Bauer for provision of some AML blast samples, and Ms C. Wartchow for critical reading of the typescript.


    1. Top of page
    2. Abstract
    4. RESULTS
    6. Acknowledgements
    7. References
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