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Molecular Biology and Genetics |
Department of Medicine, Hematology and Oncology [C. M-T., K. K., S. D., M. T., W. E. B., H. S.], and Department of Pathology [B. D-D.], University of Münster, 48129 Münster, Germany; Department of Visceral and Vascular Surgery, University of Cologne, 50924 Cologne, Germany [R. M., P. M. S.]; and Division of Hematology/Oncology, Cedars-Sinai Research Institute/University of California at Los Angeles School of Medicine, Los Angeles, California 90048 [G. I., H. P. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The important role of cyclin E has also been demonstrated by reports that cyclin E is overexpressed in several types of cancer (16, 17, 18, 19) . Immunohistochemistry studies demonstrated that high levels of cyclin E and low levels of p27 predicted a poor prognosis in young breast cancer patients (12) . In addition, cyclin E expression in cancer is often deregulated with regard to the cell cycle (20) . In two recent papers, the expression of cyclin E has been studied in lung cancer patients by immunohistochemistry (21 , 22) . The findings obtained suggested that a high labeling index for cyclin E protein might be associated with a poorer prognosis for patients with early-stage NSCLC (21 , 22) . However, there are no data regarding the important question of whether this effect is confined to cyclin E, or whether expression levels of other cdk2-associated cyclins are associated with a poor prognosis in NSCLC as well.
Recently, a new mammalian cyclin, cyclin E2, has been cloned that shows a high degree of homology to cyclin E (23 , 24) . This cyclin was expressed in lung cancer cell lines but not in their nontransformed counterparts (25) . The unknown importance of E-type cyclins in early stage NSCLC prompted us to study the level of expression and the prognostic role of cyclin E and cyclin E2 in early-stage NSCLC. Furthermore, A-type cyclins may drive cells into S phase, and levels of cyclin A2 (formerly cyclin A) are associated with the percentage of tumor cells in S phase (26) . Cyclin A1 is a recently cloned A-type cyclin that is highly expressed in acute leukemias (27) .
To study expression of the cdk2-interacting cyclins, we developed real-time quantitative RT-PCR assays that are based on the 5' nuclease assay (28 , 29) . Quantitative real-time PCR allows for an accurate quantitation of gene expression that is less error-prone and much more accurate than immunohistochemistry. In addition, quantitative RT-PCR can be performed on very small amounts of tumor tissue.
In this study, we demonstrate that expression of E-type and A-type cyclins in NSCLC is independent of tumor stage and is not associated with size or grade of the tumor. Overexpression of cyclin E and cyclin A2, but neither cyclin E2 nor cyclin A1, was found in a high percentage of tumor samples. In addition, high expression levels of cyclin E were associated with the subsequent development of metastasis and a poor prognosis. In summary, we show that in stage I and II NSCLC, cyclin E constitutes an important independent prognostic parameter for survival.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Patients with stage IIIA tumors received radiation therapy after
surgery. All patients were followed-up for a minimum period of 5 years.
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Analyses of Gene Expression by Real-time Quantitative RT-PCR.
The quantitation of cyclin mRNA levels was carried out using a
real-time fluorescence detection method (28
, 30)
. The cDNA
was prepared as described above and amplified by PCR in the ABI Prism
7700 sequence detector (PE Biosystems, Foster City, CA). The
following primers and probes were used (all 5'to 3' direction, the
sequence in the middle indicates the probe): Cyclin E: CTC CAG GAA GAG
GAA GGC AA, FAM-CGT GAC CGT TTT TTT GCA GGA TCC-TAMRA, and TCG ATT TTG
GCC ATT TCT TCA; Cyclin E2: TGT TGG CCA CCT GTA TTA TCT GG, FAM-CAC TCA
TGT TGA GAC TTA ATC CCT AAT GTG GCA-TAMRA, ATC TGG AGA AAT CAC TTG TTC
CTA TTT C; Cyclin A2: AGC TGC CTT TCA TTT AGC ACT CTA C, FAM-TCA CGG
GAC AAA GCT GGC CTG AA-TAMRA, TTA AGA CTT TCC AGG GTA TAT CCA GTC; and
PCNA: ATC ATT ACA CTA AGG GCC GAA GAT AAC, FAM- CCT TGG CGC TAG TAT TTG
AAG CAC CAA ACC-TAMRA, TCA TTT CAT AGT CTG AAA CTT TCT CCT G. The
cyclin A1 and GAPDH primers and probes have been described previously
(29)
. Primer and probe combinations were positioned to
span an exon-exon junction. When genomic DNA was used as a template, no
bands were seen after PCR amplification. The probes were labeled at the
5' end with VIC (GAPDH probe) or with FAM (all others)
and at the 3' end with TAMRA, which served as a quencher. The 5'
to 3' nuclease activity of the Taq polymerase cleaved the probe and
released the fluorescent dyes (VIC or FAM), which were detected by the
laser detector of the sequence detector (29)
. After the
detection threshold was reached, the fluorescence signal was
proportional to the amount of PCR product generated. Initial template
concentration could be calculated from the cycle number when the amount
of PCR product passed a threshold set in the exponential phase of the
PCR reaction. Relative gene expression levels were calculated using
standard curves generated by serial dilutions of U937 cDNA. The
relative amounts of gene expression were calculated by using the
expression of GAPDH as an internal standard. At least two independent
analyses were performed for each sample and for each gene. Analyses of
gene expression data were performed without the knowledge of patient
data.
Statistical data analyses were performed using SPSS 9.0 and the statistical tests indicated in "Results." The following parameters were included into the Cox regression analysis for individuals with stages I and II NSCLC: (a) cyclin E; (b) cyclin E2; (c) cyclin A1; (d) cyclin A2, (e) p53 mutation status; (f) sex; (g) smoker/nonsmoker; (h) tumor diameter; (i) histological subtype; (j) stage (I or II); (k) tumor grade; and (l) patient age. A P < 0.05 was considered as significant.
Immunohistochemistry.
Anti-cyclin E monoclonal antibody (PharMingen, San Diego, CA) was used
for immunohistochemical analysis. Frozen sections from corresponding
tumor material were cut at a thickness of 5–6 µm and mounted on
poly-L-lysine-coated glass slides and fixed in an ice-cold
methanol/aceton mix (1:1). Sections were digested with proteinase K
(0.1 µg/ml) in 1 M Tris-buffer (pH 7.5) at 37° for 5
min. Reaction was stopped in 70% ethanol, and slides were rinsed in
PBS. A 1:50 dilution of the primary antibody was applied for 45 min at
room temperature, and then a rabbit-antimouse (RPMI 1640, 1:30; 30 min
at room temperature; DAKO, Copenhagen, Denmark) and a mouse-antirabbit
phosphatase-anti-alkaline phosphatase complex (RPMI 1640, 1:100;
60 min at room temperature; Dako). Finally, the sections were rinsed in
destilled water for 10 min and counterstained with hematoxylin and
mounted in Kayser’s glycerine gelatin. Omission of the primary
antibody served as negative control. Slides were examined at x100 and
x200 by an experienced pathologist, who was not informed of the
results obtained from mRNA measurements. They were judged as
"negative," "positive," or "strongly positive," depending
on the percentage of the cells showing a nuclear staining pattern. At
least five visual fields at x200 were examined on at least two slides
per section.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Parts of the
surgically resected tumors were immediately shock-frozen and stored at
-70°C until analysis. All specimens were confirmed to contain a high
percentage of tumor cells (>70%). Lung tissue without histological
evidence of tumor infiltration was isolated from 12 of the individuals
to serve as a control. RNA preparation and reverse transcription were
carried out as described in "Material and Methods." Subsequently,
expression levels for the different cyclins and GAPDH were analyzed
using real-time quantitative RT-PCR. At least two independent analyses
of each cDNA sample were performed for each gene and comparisons of
these results showed a very high degree of reproducibility (Fig. 1A)
. For relative quantitation of gene expression, standard
curves with serial dilutions of cDNA were established (Fig. 1B)
. The amount of gene expression was standardized to
expression levels of the housekeeping gene GAPDH.
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. Levels of cyclin mRNA of tumor samples and controls are shown in Fig. 2
. Interestingly, expression of cyclin E2 was lower in most
tumor samples than in controls, and none of the tumor samples reached
the cutoff value. For cyclin A1, one control and six tumor
samples were above the cutoff value. In contrast to the low expression
levels seen for cyclin A1 and cyclin E2, a large
fraction of patients presented with elevated levels of cyclin
E (41%, 29 of 70) and cyclin A2 (31%, 22 of 70).
Cyclin E levels did not correlate significantly with the
size of the tumor or the proliferating fraction of the tumor as
indicated by PCNA expression (Table 3)
. Cyclin A2 levels were significantly higher in tumor
samples than in controls (Fig. 2)
. In contrast with cyclin
E, a close association between cyclin A2 expression and
PCNA was noted (Table 3)
.
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To analyze the relationship between cyclin expression and prognosis,
patients were grouped according to high and low expression. For
cyclin E and cyclin A2, the cutoff values as
defined in Table 2
were used. Concerning cyclin A1 and
cyclin E2, because few or none of the samples showed
increased expression, the tumor samples were split into two groups at
the median of the expression level.
Patients with low levels of cyclin E at diagnosis showed a
mean survival time of 71.7 months (95%CI 60.2–83.2 months), whereas
the mean survival time for patients with high cyclin E
levels was only 47.7 months (95%CI 37.6–57.8 months). This difference
was statistically significant (P = 0.03, log
rank test; Fig. 3A
). Differences in the expression levels of either
cyclin E2, cyclin A1, or cyclin A2 did
not predict survival (Fig. 3A)
. We further analyzed the
predictive role of low versus high cyclin E
expression in different stages of NSCLC. Strong predictive values of
cyclin E expression were detected for stage I (mean survival
59.0 versus 90.9 months) and stage II disease (mean survival
40.6 versus 74.8 months; Fig. 3B
). No significant
difference could be detected in stage IIIA patients (mean survival 30.3
versus 26.4 months).
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. The percentage of all patients who developed distant metastasis was
37.1% (26 of 70). Fewer than 25% of patients with low cyclin
E levels (10 of 41) developed distant metastasis. However, 55%
(16 of 29) of patients with high cyclin E levels developed
distant metastasis (P = 0.01). In contrast to
the predictive value of cyclin E levels, no relationship was
found between the development of metastasis and expression levels for
cyclin E2 (P = 1.0) and
cyclin A1 (P = 0.8). The small
differences seen for cyclin A2 did not reach statistical
significance (P = 0.18; Fig. 4
).
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. We found a strong association between cyclin E expression
in immunohistochemistry and the quantitative expression data obtained
by real-time RT-PCR (Fig. 6)
. The mean cyclin E mRNA expression levels of tumors without
immunohistochemistry staining (negative) was 2.52 ± 1.3
SD (n = 8), whereas mRNA expression of
positive tumors was 9.59 ± 5.7 SD
(n = 9). The differences in expression were
statistically highly significant (P = 0.01;
Mann-Whitney u-test).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To our knowledge, this is the first study to show that real-time
quantitative RT-PCR might be of clinical use for the prediction of
metastasis and survival of patients with solid tumors. Analyses of gene
expression levels can be performed at the mRNA as well as on the
protein level. The level of cyclin E protein, measured by
immunohistochemistry, has been studied previously in NSCLC (21
, 22)
. Analyses of expression levels by immunohistochemistry
reveal information about protein stability and the spatial organization
of its expression. On the other hand, immunohistochemistry usually
results in semiquantitative data only, and the reliability depends on
the antibody and detection system used and may be subject to bias.
Western blotting, another method to measure protein levels, does not
give any information about the cell type expressing the protein and is
rather difficult to perform on a large number of patient samples.
Measurements at the RNA level can overcome some of these obstacles. For
clinical specimens, analyses by RT-PCR provide a rapid way of analyzing
the presence or absence of specific transcripts. However, before the
advent of real-time PCR, the accurate measurement of the amount of
transcripts in the specimen was time-consuming and error prone. In our
study, we have used the 5' nuclease assay to determine quantitatively
the level of expression of cdk2-associated cyclins in NSCLC specimens.
Despite the convenience and accuracy of real-time PCR, several factors
have to be taken into account to avoid potential pitfalls that might
hamper the quality of the data. To minimize the problem of normal cell
contamination of the tumor sample, we only used specimens that
contained a high percentage (>70%) of tumor cells. To exclude
analysis of genomic DNA that might contaminate the RNA preparation, the
probes were designed to cover an exon-exon junction of the gene of
interest. Because pseudogenes might be present in genomic DNA, we
checked the samples for amplification in reverse transcription
reactions in the absence of reverse transcriptase. In addition, all
samples were quantitated according to a standard curve, which was run
on every PCR plate. Furthermore, the same standard samples were used
for each PCR plate to standardize results among different plates. All
samples were analyzed at least twice on independently prepared and
analyzed reaction plates. In our experience, this type of analysis
results in highly reproducible data with a high degree of correlation
of data points obtained at different occasions (Fig. 1A)
.
Because of the exponential nature of PCR, the minimum difference in
expression levels that can be detected by the 5' nuclease assay is
about 2-fold. In our analyses, we demonstrated that expression levels
of cyclins, and in particular cyclin E, vary in a wide
range. For example, high expressing samples can have more than
1000-fold higher expression than samples expressing low levels of
cyclin E. We could also demonstrate, that the cyclin
E mRNA levels measured in our assay correlated well with
immunohistochemical staining patterns of the cyclin E protein (Fig. 6)
.
The cell cycle is a basic cellular mechanism that controls fundamental processes such as growth and proliferation as well as apoptosis (31) . A large body of data evidences the pivotal role of the RB pathway in the G1-S transition and its aberrations in tumor cells (1) . Cdk2-containing complexes are critically involved in phosphorylating RB at the G1-S transition. Cyclin genes are often amplified in cancers (32) . Thus, elevated levels of cyclin E are not surprising in malignant tumors. However, it is unclear is whether cyclin E is merely reflecting the enhanced proliferation of the transformed cells, or whether elevated levels of cyclin E itself confer a more malignant phenotype. Cyclin A2, the main partner for cdk2 during S phase, was found to be elevated in a significant portion of the tumors. Its expression correlated well with levels of PCNA, suggesting that cyclin A2 expression was a consequence rather than a cause of enhanced proliferation in these tumors. We also studied expression and significance of the cdk2 partners cyclin A1 and cyclin E2, two cdk2 partners that are much less studied. Cyclin A1 is 60% identical to cyclin A2, and it enhances the cdk2 kinase activity to phosphorylate RB (33) . Its expression is regulated differently than the expression of cyclin A2 (34 , 35) . Under physiological conditions, cyclin A1 is highly expressed in testis, and it is essential for spermatogenesis (36 , 37) . In malignant disease, cyclin A1 is highly expressed in some leukemic blasts derived from myeloid lineages (27 , 37) . The homozygous deletion of cyclin A2 leads to embryonal lethality indicating that cyclin A1 does not substitute for cyclin A2 (38) . In the current study, we found increased cyclin A1 expression in six patient samples, whereas the majority of lung tumors expressed very low levels of cyclin A1. These findings show that cyclin A1 expression occurs in some solid tumors but in general appears to be a rare event.
Cyclin E2 is a recently cloned homologue of cyclin E and its expression was reported previously in several lung cancer-derived cell lines (25) . Our data show that expression of cyclin E2 in primary NSCLC is very low, and it is not associated with levels of either PCNA or cyclin E. These findings indicate that functions of cyclin E2 in the cell cycle obviously differ from those of cyclin E. In addition, its low expression in primary tumors casts doubt onto a role for this cyclin in the pathogenesis of NSCLC. The discrepancy between the prominent expression found in cell lines with primary tumors needs additional investigation.
Cyclin E was overexpressed in the majority of tumor samples studied and closely associated with survival in early-stage NSCLC. Of all of the cdk2-associated cyclins studied, cyclin E was the only one to have prognostic relevance. Inasmuch as all tumors included into our study were pathologically confirmed to be completely resected, enhanced cellular proliferation by itself would not worsen the prognosis of the patient. In addition, tumor size did not correlate with levels of cyclin E in our study. Also, cyclin E levels in our study and in a previous study (21) did not correlate with levels of PCNA; and levels of cyclin A2, which we show to be closely associated with cellular proliferation, did not predict survival. It has been shown that cyclin E expression in cancer cells can be unrelated to the phase of the cell cycle (39) . A possible explanation for the strong prognostic value of cyclin E may be suggested by our finding that elevated levels of cyclin E were closely associated with the development of distant metastasis. How could cyclin E be linked to metastasis? One possibility is that targets of cyclin E-cdk2 complexes are involved in the loss of cellular adherence, and the increased cellular mobility associated with mitosis strengthens this point of view. Another possible explanation is conveyed by recent findings that overexpression of cyclin E leads to increased chromosome instability (40) . High levels of cyclin E lead to impaired S phase progression and the development of aneuploidy in the cell population. This phenomenon was observed solely when cyclin E, but not when cyclin A2 or cyclin D1, were overexpressed. These findings in cell lines correspond well with the unique prognostic role of cyclin E levels in our study population. The induction of chromosomal instability by deregulation of cyclin E might be an important mechanism in the development of metastasis in early-stage NSCLC. To analyze this possibility in more detail, we used comparative genomic hybridization to examine overall genomic instability in high- and low-cyclin E-expressing tumors. However, we did not detect significant differences (data not shown). An alternative hypothesis might be that cyclin E is simply an indicator gene and does not have pathogenetic relevance. Even if this is the case, analyses of cyclin E expression levels might be clinically useful as an indicator for the likelihood of metastasis in NSCLC. This information may help to choose patients who are most likely to profit from adjuvant or neoadjuvant chemotherapeutic treatment strategies in the early stages of the disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Deutsche Krebshilfe
(10-1539-Mü1), the Deutsche Forschungsgemeinschaft (Mu 1328/2-1,
Se 600/2-2), the IMF-Program (Mü429926, Mü529905, and
SE119908) at the University of Münster, and the University of
California Tobacco Related Disease Program. This study is part of
Katrin Kügler’s M. D. thesis work. 
2 To whom requests for reprints should be
addressed, at: Department of Medicine, Hematology and Oncology,
University of Münster, Domagkstr. 3, 48129 Münster,
Germany. Phone: 49-251-835-6229; Fax: 49-251-835-2673; E-mail: muellerc{at}unimuenster.de
3 RB, retinoblastoma gene product; cdk,
cyclin-dependent kinase; NSCLC, non-small cell lung cancer; RT-PCR,
reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; TAMRA, 6-carboxytetramethylrhodamine; PCNA,
proliferating cell nuclear antigen.
Received 3/24/00. Accepted 11/ 8/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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