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Keywords:

  • acute myeloid leukaemia;
  • prognosis;
  • cyclin A1;
  • cyclin A (A2);
  • cell cycle

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Expression of cyclin A (A2) and A1 mRNA in AML
  6. Relationship between clinical data and expression of cyclin A1 and A (A2)
  7. Expression levels of cyclin A (A2) and A1 and survival of AML patients
  8. Discussion
  9. Acknowledgments
  10. References

Summary. Cyclin A (A2) and cyclin A1 are members of the G2 cyclins, which are involved in the control of G2/M and G1/S transitions as well as mitosis. Human cyclin A1 was cloned as an A-type cyclin that is highly expressed in acute myeloid leukaemia (AML). The clinical significance of these cyclins in myeloid leukaemia remains to be clarified. We investigated the relative levels of these transcripts in 80 patients with de novo AML. Correlations with clinical parameters showed that the initial white blood cell count and serum lactate dehydrogenase levels were inversely associated with cyclin A (A2) mRNA levels (r = −0·276, P = 0·019) and cyclin A1 mRNA levels (r = −0·241, P = 0·042) respectively. They were independently associated with increased overall survival [P = 0·035 for cyclin A (A2) and P = 0·016 for cyclin A1]. Multivariate analysis using Cox's proportional hazard model showed that elevated cyclin A1 mRNA levels contributed significantly to the better prognosis of patients with AML. Furthermore, the analysis of survival probability showed that the group with high levels of both cyclin A (A2) and A1 survived significantly longer than the group with low expression of both these cyclins (P = 0·002). These data indicate that high expression levels of both cyclin A (A2) and A1 are associated with good prognosis in AML patients.

The regulation of the cell cycle is of particular importance for the haematopoietic system; dysregulation of the cell cycle machinery may contribute to the development of haematopoietic malignancies as well as other types of cancers (Sherr, 1996). Critical components of the basic cell cycle regulation have been identified including cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs) (Pines, 1993; Sherr & Roberts, 1999). Proteins involved in cell cycle regulation are also important as therapeutic targets in haematopoietic malignancies (Broxmeyer, 1995; Afenya, 1996). Several studies have found correlations between the cell cycle characteristics of acute myeloid leukaemia (AML) cells and their response to cytotoxic chemotherapy and/or their clinical outcome (Boiron et al, 1994; Lacombe et al, 1994).

Cyclin A (A2) is a member of the G2 cyclins that are involved in the control of the G2/M cell cycle transition and mitosis, as well as S-phase progression (Pagano et al, 1992; Zindy et al, 1992). A previous study indicated that expression of cyclin A (A2) mRNA was a marker of cell proliferation in several human haematological malignancies, showing a highly significant correlation between expression of either cyclin A (A2) mRNA or protein and the cumulative percentage of cells in S plus G2/M phase (Paterlini et al, 1993). Cyclin A (A2) may be associated with the chemosensitivity of leukaemic blast cells for the following reasons: (1) expression levels of cyclin A (A2) mRNA have a tendency to decrease after relapse compared with the primary leukaemia; (2) expression levels of cyclin A (A2) have a positive correlation with the expression of topoisomerase II mRNA; (3) expression levels of cyclin A (A2) have an inverse correlation with multidrug resistance 1 (mdr1) RNA expression, and elevated levels of the latter are characteristic of refractory acute leukaemia cells (Beck et al, 1995, 1996).

Human cyclin A1 is an A-type cyclin that is highly expressed only in the testes in healthy tissues (Yang et al, 1997). It is essential for meiosis in murine spermatogenesis (Liu et al, 1998). In vitro experiments have shown that several functional similarities are shared between cyclin A (A2) and cyclin A1 in cell cycle regulation, including interaction with E2F-1, p107 and p130 in an analogous manner and ability to reverse a G1 arrest induced by retinoblastoma (Rb) protein in SAOS-2 cells (Yang et al, 1999a). Interestingly, human cyclin A1 is highly expressed in several types of AML (Yang et al, 1997, 1999b). Recently, AML was reported to develop in transgenic mice that overexpressed cyclin A1 (Liao et al, 2001). Various AMLs have been associated with high levels of C-MYB (Muller et al, 1999), PML-RAR or PLZF-RAR (Muller et al, 2000), and these proteins are known transcriptionally to enhance the expression of cyclin A1. The role of cyclin A1 in the mitotic cell cycle and the significance of its overexpression in myeloid leukaemia cells remain to be clarified.

Levels of cyclins A1 and/or A (A2) in leukaemia cells may have significance concerning their chemosensitivity as well as the overall prognosis of the patient. In the present investigation, we studied the expression levels of cyclin A (A2) and A1 mRNA in 74 de novo AML patients, who were treated by standard chemotherapy protocol based on daunorubicin and a conventional dose of cytarabine arabinoside (Ara-C), except for those with acute promyelocytic leukaemia (APL; M3) who received all-trans retinoic acid (ATRA) either with or without chemotherapy. The data were analysed to determine whether cyclin A (A2) and A1 levels have predictive value for disease prognosis. We found that high levels of expression of both cyclins A and A1 contributed to better overall survival of de novo AML patients.

Clinical samples.  Bone marrow (BM) and peripheral blood (PB) samples from 80 patients with newly diagnosed AML were obtained with their informed consent at onset, before chemotherapy. AML was classified according to the criteria revised by the French–American–British (FAB) classification (Bennett et al, 1985). AML patients were treated with Ara-C (or behenoyl Ara-C), daunorubicin (or idarubicin), either with or without prednisolone and/or 6-mercaptopurine. Complete remission (CR) was defined as a BM aspirate that showed trilineage regeneration with less than 5% blasts by morphological and immunochemical analyses, in the presence of a normal blood count that persisted for at least 1 month (Cheson et al, 1990). Patients who died of toxic complications (infection and bleeding) before the time of expected marrow recovery were not evaluated. All other patients were considered as non-responsive (NR). To purify leukaemia cells, heparinized PB cells or BM aspirates were mixed with an equal volume of Roswell Park Memorial Institute (RPMI)-1640 medium and centrifuged on Ficoll–Hypaque (Pharmacia, Uppsala, Sweden). Normal BM and PB cells were obtained from healthy volunteers after obtaining their informed consent. Total RNA was extracted as described previously using guanidium thiocyanate (Chomczynski & Sacchi, 1987).

Reverse transcription polymerase chain reaction (RT-PCR).  Complementary DNA (cDNA) was made using a Superscript cDNA synthesis kit (Gibco BRL, Rockville, MD, USA). The RNA (0·6 µg) was reverse transcribed to synthesize cDNA using random primers at 42°C, then 1 µl of the cDNA product was amplified by PCR using specific primers (0·5 µmol/l) in 50 µl of mixture consisting of 10 mmol/l Tris-HCl (pH 8·3), 50 mmol/l KCl, 1·5 mmol/l MgCl2 and 0·5 mmol/l dNTPs (dATP, dTTP, dGTP, dCTP). The oligonucleotides used in the PCR amplification were as follows: sense strand, 5′-GCCTGGCAAACTATACTGTG-3′; antisense strand, 5′-CTCCATGAGGGACACACACA-3′ for human cyclin A1; sense strand, 5′-TCCATGTCAGTGCTGAGAGGC-3′; antisense strand 5′-GAAGGTCCATGAGACAAGGC-3′ for human cyclin A (human cyclin A2); sense strand, 5′-CAGCCGAGCCACATCG-3′; antisense strand, 5′-TGAGGCTGTTGTCATACTTCTC-3′ for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Based on the sequence information surrounding the intron–exon junctions of each gene, the primers were designed to span an intron; therefore, the PCR product specifically detects mRNA. PCR comprised 19 cycles for cyclins A1 and A and 13 cycles for GAPDH, with denaturing at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 30 s. The reaction was performed in a GeneAmp PCR system 9600 (Perkin-Elmer, Norwalk, CT, USA). The semi-quantitative RT-PCR was performed as described previously (Yang et al, 1999b; Kawabata et al, 2001). The PCR products were subjected to 1·5% agarose gel electrophoresis and transferred to Hybond-N+ membrane (Amersham) by the capillary method. After the transfer, the membrane was hybridized with each cDNA probe labelled with digoxigenin labelling systems (Boehringer Mannheim, Indianapolis, IN, USA). The signal intensity of each specific band was evaluated by means of a Fluor-S Multimager (Bio-Rad Laboratories, Richmond, CA, USA). The linearity of the semi-quantitative RT-PCR products was determined using various amounts of total RNA obtained from human myeloid leukaemia ML1 cells. Under these conditions, the signal intensity of the PCR products increased proportionally up to 1 µg of total RNA (data not shown).

Statistical analysis.  Statistical comparisons between groups were performed by means of Mann–Whitney's U-test (non-parametric analysis), and P < 0·05 indicated a significant difference. Pearson's correlation was used to evaluate the correlation between paired values. Survival curves of patients were prepared by the Kaplan–Meier method, and differences between the survival curves were evaluated using log-rank, generalized Wilcoxon's tests and Cox–Mantel tests. A multivariate analysis of the prognosis was performed using Cox's proportional hazard model.

Expression of cyclin A (A2) and A1 mRNA in AML

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Expression of cyclin A (A2) and A1 mRNA in AML
  6. Relationship between clinical data and expression of cyclin A1 and A (A2)
  7. Expression levels of cyclin A (A2) and A1 and survival of AML patients
  8. Discussion
  9. Acknowledgments
  10. References

To normalize for the differences in RNA loading for RT-PCR and RNA degradation in individual samples, the signal intensities of the cyclin A1 and A (A2) mRNA were divided by those from the GAPDH mRNA, and each of these was compared with the value derived from ML1 mRNA, which was defined as 100 (the expression index). Expression of cyclin A1 and A (A2) genes was also analysed by quantitative RT-PCR in mononuclear cells from PB and BM of normal individuals as additional controls. The normal samples expressed much less of both cyclin A1 and A (A2) mRNAs compared with ML1 myeloid leukaemia cells. Expression of cyclin A (A2) mRNA in normal BM cells was significantly higher than in normal PB cells (P = 0·028), whereas expression of cyclin A1 mRNA in normal BM cells was slightly lower than that in normal PB cells (P = 0·601).

A summary of the statistical data from the normal haematopoietic and AML cells is given in Table I. Eighty cases were studied including six APL (M3) samples. The levels of mRNA expression of both cyclin A1 and A (A2) genes in AML cells was significantly higher than those in normal cells, which included both PB and BM mononuclear cells (P < 0·01, Table I). Levels of cyclin A1 mRNA were statistically different compared with normal cells, which included both PB and BM mononuclear cells, and leukaemic blasts from FAB-M1, M2, M3 and M5 (Table I). Likewise, levels of cyclin A (A2) differed significantly between normal cells and blast cells from FAB-M1, M2 and M5 (Table I). However, no significant differences were found in expression levels of cyclin A (A2) between BM mononuclear cells from normal individuals and blast cells from any FAB subtypes of AML (Table I). As we reported previously (Yang et al, 1999b), the expression level of cyclin A1 in AML-M3 leukaemic cells (index 120 ± 73) was significantly higher than that of leukaemia cells from AML-M2-M6 subtypes (index 61 ± 58; P = 0·038). In contrast, the level of cyclin A (A2) mRNA in AML-M3 was significantly lower (index 19 ± 5) than levels in the AML cells of other AML subtypes (index 93 ± 155; P = 0·043,Table I). No statistical correlation was found between levels of mRNA expression between cyclin A1 and A (A2) in the AML cells (r = 0·006, P = 0·957 by Spearman correlation coefficient). Statistical significance could not be analysed for AML-M0 (n = 2) and M6 (n = 1) because of the small number of cases investigated.

Table I.  Levels of cyclin A1 and cyclin A (A2) mRNA in normal and AML cells.
FAB typeNo. of casesCyclin A1P versus NormalCyclin A (A2)P versus Normal
  • The mRNA levels were normalized for GAPDH mRNA.

  • The positive control (index = 100) is represented by RNA extracted from the ML1 acute myeloblastic leukaemia cell line. Analysed by means of Mann–Whitney test (versus normal).

  • Normal, normal cells that include both peripheral blood and bone marrow mononuclear cells.

  • PB, peripheral blood mononuclear cells.

  • §

    BM, bone marrow mononuclear cells.

  • AML, acute myeloid leukaemia.

  • *

    P  < 0·05;

  • * *

    P  < 0·01;

  • * * *

    P  < 0·001.

Normal (PB)521 ± 8 10 ± 4 
Normal (BM§)518 ± 8 23 ± 8 
M0217 ± 4 88 ± 114 
M120102 ± 87< 0·001***118 ± 181< 0·05*
M22171 ± 58< 0·01**68 ± 59< 0·01**
M36120 ± 73< 0·01**19 ± 50·355
M41244 ± 350·098136 ± 280·055
M51871 ± 70< 0·05*61 ± 69< 0·05*
M61  10 165 
AML (except M3)7472 ± 69< 0·01**93 ± 155< 0·01**
Total AML8076 ± 70< 0·01**86 ± 151< 0·01**

Relationship between clinical data and expression of cyclin A1 and A (A2)

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Expression of cyclin A (A2) and A1 mRNA in AML
  6. Relationship between clinical data and expression of cyclin A1 and A (A2)
  7. Expression levels of cyclin A (A2) and A1 and survival of AML patients
  8. Discussion
  9. Acknowledgments
  10. References

We investigated the associations between expression levels of cyclin A1 and A (A2) mRNA and age, gender, white blood cell (WBC) count, lactate dehydrogenase (LDH), cytogenetics and response to initial therapy in 74 AML cases, excluding APL (M3). The clinical background and the cyclin A1 and A (A2) mRNA levels of these patients are summarized in Table II. No significant difference in expression levels of cyclin A1 occurred between the groups based on gender and age, whereas a significant inverse correlation was noted between age and cyclin A (A2) expression (Table II; r = −0·232, P = 0·049 by Spearman correlation coefficient). Also, elevated WBC count and LDH were inversely correlated with expression of cyclin A (A2) (r = −0·276, P = 0·019 by Spearman correlation coefficient) and A1 mRNA(r = −0·241, P = 0·042 by Spearman correlation coefficient) respectively. These correlations were weak, but significant (Table II). No apparent association was found between expression of either cyclin A (A2) or A1 mRNA and either specific cytogenetics or response to initial chemotherapy.

Table II.  Relationship between cyclin A1 and cyclin A (A2) expression and clinicopathological features.
Clinical factors (No.)Cyclin A1 indexPCyclin A (A2) indexP
  • CR, complete remission.

  • NR, non-responsive.

  • Values are mean ± SD.

  • Analysed by Mann–Whitney U-test. *P < 0·05.

Age (years) ≤  64(45)74 ± 64 118 ± 188 
> 65(29)69 ± 760·48853 ± 650·0434*
SexM(39)72 ± 70 97 ± 147 
F(35)73 ± 690·96989 ± 1660·973
WBC (× 109/l) ≤  10(28)82 ± 79 86 ± 73 
> 10, ≤ 50(29)77 ± 710·959133 ± 2330·533
> 50(17)48 ± 380·15639 ± 490·014*
LDHNormal (N)(12)108 ± 99 152 ± 226 
> N, ≤ 5 × N(56)64 ± 540·285 ± 1420·647
> 5 × N(5)32 ± 200·026*53 ± 890·139
Cytogenetic abnormalities(40)67 ± 68 110 ± 202 
+(34)79 ± 710·38573 ± 710·678
t(8;21)(8)67 ± 440·54286 ± 720·335
11q23(3)111 ± 1130·390118 ± 1590·980
Others(23)78 ± 740·56763 ± 560·965
Response to initial chemotherapyCR(46)72 ± 69 114 ± 187 
NR(27)73 ± 700·91557 ± 650·185

Expression levels of cyclin A (A2) and A1 and survival of AML patients

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Expression of cyclin A (A2) and A1 mRNA in AML
  6. Relationship between clinical data and expression of cyclin A1 and A (A2)
  7. Expression levels of cyclin A (A2) and A1 and survival of AML patients
  8. Discussion
  9. Acknowledgments
  10. References

We examined the relationship between the level of cyclin A1 and/or A (A2) mRNA and overall survival of the AML patients. The 74 AML patients were divided into two groups based on their levels of expression of cyclin A (A2) mRNA. Those with high levels of cyclin A (A2) gene expression (above 75) had a significantly higher rate of overall survival (25 patients, P = 0·035, generalized Wilcoxon's test; P < 0·05, log-rank test) than those (48 patients) with levels below 75 (Fig 1B and Table III). However, if survival was compared with either the mean or mean + 2SD of cyclin A (A2) levels as the cut-off value, no significant prognostic correlates were noted (data not shown). In contrast, all the cut-off values including either mean levels or mean + 2SD showed prognostic significance in the case of cyclin A1, and the results using 65 as a cut-off value gave the best separation of good and bad prognosis (P = 0·016, generalized Wilcoxon's test; P < 0·05, log-rank test; Fig 1A and Table III).

image

Figure 1. (A) Survival curves of AML patients. Patients whose blast cells expressed high cyclin A1 (index > 65) (n = 30, solid line) had a better prognosis than those with low cyclin A1-expressing blast cells (index < 65) (n = 40, dotted line) (generalized Wilcoxon's test, P = 0·016 and log-rank test, P < 0·05). (B) Survival curves of AML patients. High cyclin A (A2) (index > 75) patients (n = 25, solid line) had a better prognosis than those with low cyclin A (A2) (index < 75) (n = 45, dotted line) (generalized Wilcoxon's test, P = 0·035 and log-rank test, P < 0·05). (C) Survival curves of AML patients. Comparison of survival curves of groups A, B, C and D. Groups A, B, C and D are described in Table III.

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Table III.  Comparisons of survival probability between the four groups as subdivided by the level of expression of cyclin A1 and A (A2).
Groups*Generalized Wilcoxon (P)Cox–Mantel (P)Log rank (P)
  • *

    Group A, cyclin A (A2) and A1 both high; group B, A (A2) and A1 both low; group C, A (A2) low, A1 high; group D, A (A2) high, A1 low.

  • NS, not significant.

Group A versus B0·002< 0·01< 0·01
Group A versus CNSNSNS
Group A versus D0·05NSNS
Group B versus CNS0·038NS
Group B versus DNSNSNS
Group C versus DNSNSNS
Group A versus (B, C, D)0·0090·017< 0·05
Group (A, C) versus (B, D)0·0160·012< 0·05
Group (A, D) versus (B, C)0·0350·015< 0·05

To understand the relationship between expression levels of cyclin A (A2) and A1 in the various AML cases, these values were plotted and divided into four groups. As described above, the cut-off numbers for the expression indices of cyclin A1 and A (A2) were 65 and 75 respectively. Both cyclin A1 and A (A2) levels were high in group A; both were low in group B; only cyclin A1 was high in group C; and only cyclin A (A2) was high in group D. Twenty-eight cases were in group B (35%), 27 individuals in group C (34%), 16 cases in group D (20%) and nine patients in group A (11%) (Table IV). Four of the six AML-M3 cases (67%) were categorized as group C (Table IV), and the other two cases were in group B, reflecting the relatively high expression levels of cyclin A1 (expression indices were above 50; data not shown) in APL.

Table IV.  Classification of AML patients by their levels of cyclin A1 and cyclin A (A2) mRNA.
FABNo. of patientsGroup*
ABCD
  • According to the levels of cyclin A1 and cyclin A (A2), 80 AML patients were divided into four groups.

  • *

    Group A, cyclin A (A2) and A1 both high; group B, A (A2) and A1 both low; group C, A (A2) low, A1 high; group D, A (A2) high, A1 low.

  • CR, complete remission.

  • NR, non-responsive.

  • Numbers in parentheses are percentages.

M020101
M12021107
M2213873
M360240
M4121632
M51831032
M61   1
AML (except M3)749 (12)26 (35)23 (31)16 (22)
Total AML809 (11)28 (35)27 (34)16 (20)
CR 7/9 (78)14/25 (56)14/23 (61)12/16 (75)
NR 2/9 (22)11/25 (44)9/23 (39)4/16 (25)

We found an inverse correlation between cyclin A1 and serum LDH levels, as well as an inverse correlation between cyclin A (A2) and initial WBC in 74 cases, with the exception of the APL (FAB, M3) cases. As the WBC has been reported to be a negative prognostic factor in AML patients (Kantarjian et al, 1988), we analysed the association of the cyclin A1 and A (A2) mRNA expression levels of these patients with their chemosensitivity and probability of survival. The CR ratios of groups A, B, C and D were 78%, 56%, 61% and 75% respectively (Table IV). The group with higher expression levels of cyclin A (A2) (groups A + D) showed a higher CR ratio (76%) than those with higher expression levels of cyclin A1 (groups A + C, 68%). Although no statistically significant difference was noted between CR ratios and levels of expression of either cyclin A1 or A (A2) mRNA (Table II), the sensitivity to initial chemotherapy tended to be more closely associated with the levels of cyclin A (A2) expression than with those of cyclin A1.

Next, we compared the survival probability between the four groups: group A versus B, A versus C, A versus D, B versus C, B versus D and C versus D. Among these four groups, group A [high A1•high A (A2)] had the longest survival times, and group B [low A1•low A (A2)] exhibited the shortest survival times (Fig 1C). Group A survived significantly longer than either group B (P = 0·002, generalized Wilcoxon's test; P < 0·01, log-rank test) or groups B + C + D (P = 0·009, generalized Wilcoxon's test; P < 0·05, log-rank test) (Fig 1C, Table III). Although the comparisons between all the other groups indicated no significant difference in survival by log-rank test, the duration of survival of those in group A [high A1•high A (A2)] was statistically longer than those in group D [low A1•high A (A2)] (P = 0·05, generalized Wilcoxon's test), and the survival time of group C [high A1•low A (A2)] was statistically longer than that of group B [low A1•low A (A2)] (P = 0·038, Cox–Mantel test) (Fig 1C and Table III), suggesting that elevated expression of cyclin A1 is more closely associated with a good prognosis compared with increased levels of cyclin A (A2) in AML patients. Cox's proportional hazard model was used to evaluate the relative importance of cyclin A1 and cyclin A (A2) as putative prognostic factors in 74 AML patients in the present study. As shown in Table V, we found that expression levels of cyclin A1 mRNA were a significant prognostic factor next to age among factors including age, gender, cyclin A1, cyclin A (A2), WBC count, LDH level and cytogenetic group with favourable prognosis [t(8;21) and inv16] (Grimwade et al, 1998). These results showed that the level of cyclin A1 gene expression represents a new prognostic factor for AML. The haematological characteristics and outcomes of all the patients in group A are shown in Table VI.

Table V.  Multivariate analysis of clinical factors on overall AML patient survival *.
VariableP
  • *

    Excludes type M3 cases, who receive a unique therapy, including all-trans retinoic acid (ATRA) either with or without chemotherapy.

Age (≤ 64/> 65)0·003
Sex (M/F)0·718
Cyclin A10·012
Cyclin A (A2)0·170
WBC (≤ 50/> 50 × 109/l)0·384
LDH (≤ N, 5 × N/> 5 × N)0·446
t(8;21), inv(16)0·117
Table VI.  Haematological characteristics and outcome of patients in group A.
Cyclin A (A2)* Cyclin A1*FAB Age/sexWBC (× 109/l) LDHKaryotypeResponseSurvival (months)
  • *

    The positive control (index = 100) is represented by RNA extracted from the ML1 cell line.

  • LDH is represented by the ratio to the upper limit of normal range.

  • NR, non-responsive.

  • §

    CR, complete remission.

  • (Down), Down's syndrome.

10984M5b66/F10·81·346,XYNR2+
33484M429/M43·11·546,XYCR§24
106102M5a20/M4·21·146,XY, +21(Down)CR10·1
130105M248/F35·32·646,XXCR31+
168129M258/F0·41·246,XXCR22·3+
118141M273/F2·41·546,XX,t(8;21)CR66+
105224M5a24/M39·52·546,XYNR12·2+
 79234M127/M9·10·846,XYCR79·8+
136305M169/F3·40·746,XYCR29·6+

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Expression of cyclin A (A2) and A1 mRNA in AML
  6. Relationship between clinical data and expression of cyclin A1 and A (A2)
  7. Expression levels of cyclin A (A2) and A1 and survival of AML patients
  8. Discussion
  9. Acknowledgments
  10. References

The present results show an inverse relationship between the expression of both cyclins A (A2) and A1 and clinical parameters representing the leukaemic cell mass, such as initial leucocyte counts and serum LDH level, as well as a direct correlation between a better prognosis and higher expression of these cyclins. These results are somewhat counterintuitive considering that these cyclins may enhance cellular proliferation by accelerating entry into S phase (Resnitzky et al, 1995). However, the present results partly agree with a previous finding that decreased expression of cyclin A (A2) mRNA is associated with multidrug resistance of the AML cells (Beck et al, 1996).

The detailed implications of the overexpression of cyclin A (A2) and A1 in myeloid leukaemia cells in vivo are still unknown. Experimental studies have shown that cyclin A1, as well as cyclin A, efficiently bound to E2F-1 (Yang et al, 1999a). Perhaps CDK2-cyclin A (A2) and/or A1 complexes counteract the growth-promoting effects of E2F-1 by decreasing the ability to bind DNA and transactivate target genes and, as a result, exhibit negative regulation for S-phase progression (Krek et al, 1994, 1995). Consistent with this potential activity, expression of cyclin A1 and cyclin A (A2) was inversely correlated with clinical parameters associated with leukaemic cell proliferation such as WBC count and serum LDH level.

In accordance with a previous study (Paterlini et al, 1993), Muller-Tidow et al (2001) found a close association between DNA replication of cancer cells as measured by PCNA expression and levels of cyclin A (A2) in these cells. Although less significant, a similar association was noted in the case of cyclin A1 (Muller-Tidow et al, 2001). Perhaps this overexpression contributes to the increased chemosensitivity of the myeloid leukaemia cells, by stimulating these cells into S phase of the cell cycle (Miyauchi et al, 1989; Lacombe et al, 1994; Bai et al, 1999). In fact, although not statistically significant, the response rate to initial chemotherapy had a tendency to increase in the cohort with highly expressed cyclin A (A2) (Table II), and the CR ratio was highest in group A [high cyclin A (A2)•high cyclin A1] (Table IV).

Alternatively, overexpression of cyclin A (A2) and/or A1 may predispose leukaemic cells to undergo apoptosis through deregulated progression to the S or M phase of the cell cycle. This hypothesis is supported by experimental data showing that cyclin A (A2)–CDK activation and/or increased expression of cyclin A (A2) can, in some situations, induce apoptosis of tumour cells (Hoang et al, 1994; Meikrantz et al, 1994; Bortner & Rosenberg, 1995; Shi et al, 1996). In addition, a recent study also indicated that the activation of cyclin A1–CDK2 is involved in the induction of apoptosis of somatic cells (Anderson et al, 1997).

The poor response to therapy in the elderly individual with AML has been associated with an unfavourable cytogenetic change, as well as the expression of MDR1 and CD34 in the blast cells (Leith et al, 1997). In addition, in the elderly patient, AML cells have been associated with a resistance to apoptosis (Garrido et al, 2001). In this context, the lower expression of cyclin A (A2) mRNA in the AML blasts of the elderly, as shown in the present study, may contribute to their chemoresistance.

In vitro studies have found a similar functional role of cyclin A1 and A (A2) in the progression of the cell cycle (Hinds et al, 1992; Yang et al, 1999a). However, the increased sensitivity to chemotherapeutic drugs and/or apoptotic stimuli through the accumulation of S/G2M of the cell cycle of leukaemia cells is unlikely to be the sole explanation for elevated levels of both cyclin A1 and cyclin A (A2) contributing to a better prognosis. Elevated expression of cyclin A1 mRNA rather than cyclin A (A2) appears to be a better prognostic factor, as shown in the present study. Nevertheless, both these cyclins have been reported to be significantly associated with DNA replication in cancer cells (Muller-Tidow et al, 2001).

A highly significant relationship has been reported between remission duration and cell cycle time (Tc) as well as between remission duration and length of the S phase (Ts) of the cell cycle in AML (Raza et al, 1990). Although precise data were not shown, in vitro fluorescence-activated cell sorting/cell cycle analysis of bone marrow cells from cyclin A1-overexpressing transgenic mice had fewer cells in the G2/M phases of the cell cycle compared with controls (Liao et al, 2001). Overexpression of cyclin A1 and/or A (A2) might prolong Ts and Tc in vivo, and thus contribute to a better overall survival in AML patients. For example, leukaemic cells from AML-M3 patients have a significantly prolonged Tc (Raza et al, 1990) and very high levels of cyclin A1.

Recent studies have focused mainly on the alterations in the G1 regulators in the development of haematological malignancies as well as other types of cancer (Malumbres & Barbacid, 2001). Cyclin A (A2), and possibly cyclin A1, may play an important role in the signalling pathways regulating cellular proliferation (transformation) and apoptosis in AML (Meikrantz & Schlegel, 1996; Adachi et al, 2001).

Recent genome-wide cDNA microarray analysis has identified genes involved in the sensitivity of AML cells to chemotherapy (Okutsu et al, 2002). Although some cell cycle-related genes, such as PCNA and E2F1, were found to be overexpressed in poor responders to chemotherapy, alterations in expression of many cell cycle-related genes, including cyclin A1 and cyclin A (A2) as well as cyclin E (Iida et al, 1997), p27 (Yokozawa et al, 2000) and p15 (Quesnel et al, 1998), have not been identified. Nevertheless, other studies found that altered levels of these genes were related to the prognosis of AML. A larger combined study that includes cDNA microarray analysis, real-time quantitative RT-PCR analysis and possibly protein analysis will be required to evaluate the prognostic factors in AML comprehensively.

The present results found that the expression levels of both cyclin A (A2) and A1 are useful indicators of prognosis for individuals with AML. The clinical significance of expression of these cyclins in other haematological malignancies remains to be investigated, and further studies are required to understand the precise mechanisms by which these cyclins influence the prognosis of AML patients.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Expression of cyclin A (A2) and A1 mRNA in AML
  6. Relationship between clinical data and expression of cyclin A1 and A (A2)
  7. Expression levels of cyclin A (A2) and A1 and survival of AML patients
  8. Discussion
  9. Acknowledgments
  10. References

We are grateful for the generous support of the Parker Hughes Fund, J. Troy Trust and Ko-So Foundation; H.P.K. is a member of the Jonsson Comprehensive Cancer Center and Molecular Biology Institute of UCLA and holds the endowed Mark Goodson Chair of Oncology Research at Cedars-Sinai Medical Center, UCLA School of Medicine. We thank A. Nagasawa (Showa University School of Medicine) for technical assistance.

References

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  2. Abstract
  3. Patients and methods
  4. Results
  5. Expression of cyclin A (A2) and A1 mRNA in AML
  6. Relationship between clinical data and expression of cyclin A1 and A (A2)
  7. Expression levels of cyclin A (A2) and A1 and survival of AML patients
  8. Discussion
  9. Acknowledgments
  10. References
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