Differential expression of DNA topoisomerases in non-small cell lung cancer and normal lung

BB et Biophysica

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Biochimica et Biophysica Acta 1264 (1995) 337-346

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Differential expression of DNA topoisomerases in non-small cell lung cancer and normal lung Giuseppe Giaccone a,*, Jannette van Ark-Otte a, Giorgio Scagliotti b, Giovanni Capranico c, Paul van der Valk a, Gonzalo Rubio a, Otilia Dalesio a.l, Rafael Lopez a,2, Franco Zunino c, Jan Walboomers a, Herbert M. Pinedo a a Free Unit'ersity Hospital, Department of Oncology and Pathology, HV 1081 Amsterdam, The Netherlands b University of Turin, Department of Clinical and Biological Sciences, 10100 Orbassano, Italy c lstituto Nazionale per Io Studio e la Cura dei Tumori, Experimental Oncology B, 20133 Milan. Italy

Received 1 May 1995; revised 11 July 1995; accepted 10 August 1995

Abstract DNA topoisomerases are ubiquitous nuclear enzymes, and important targets of cancer chemotherapy. Expression of topoisomerase genes is often correlated with in vitro chemosensitivity. We investigated the expression of the topoisomerase genes in normal lung and non-small cell lung cancer. Expression of topoisomerase II-a, topoisomerase II-/3, and topoisomerase I genes has been assessed in tumor samples of 60 patients who underwent operation for a non-small cell lung carcinoma, by RNase protection assay, and by immunohistochemistry. The expression of topoisomerase II-a gene was either undetectable or very low in normal lung, while most NSCLC expressed readily quantifiable levels of this gene. No alteration of the topoisomerase II-c~ gene was found by Southern blotting in the NSCLC samples. In contrast to topoisomerase II-a, topoisomerase II-/3 was expressed in most normal as well as in tumor tissue samples, at a similar level. The levels of expression of both topoisomerase II isoforms was lower than that of human lung cancer cell lines. The results of the topoisomerase I! mRNA expression were confirmed by immunohistochemistry. Whereas topoisomerase II-a staining was mainly limited to the nucleus, staining with topoisomerase II-/3 antibody was exclusively observed in nucleoli. Topoisomerase I was localized in the nuclei and expression was mainly limited to tumor cells. By RNase protection, topoisomerase I expression in NSCLC samples was in the range of that of human lung cancer cell lines. The expression of the topoisomerase genes did not seem to be coordinated. In tumor cells, there was a positive association between expression of topoisomerase II-a and Ki-67, a marker of cell proliferation, as assessed by immunohistochemistry, but not with topoisomerase II-/3 or topoisomerase I. Clinical characteristics of the patients, and their survival did not appear to be correlated to the level of expression of any of the topoisomerase genes, although a trend towards a shorter survival was observed in patients whose tumors expressed relatively high topoisomerase II-a mRNA levels. In conclusion: (1) the two isoforms of topoisomerase II are differentially expressed in normal lung and NSCLC cells; (2) higher topoisomerase II-a expression is associated with higher cell proliferation in NSCLC; (3) the expression of topoisomerase II-a and topoisomerase I, but not of topoisomerase II-fl, was higher in tumor cells compared to normal lung. Given the differential expression of topoisomerases in normal lung and tumors, research of more potent and specific topoisomerase inhibitors might prove beneficial in non-small cell lung cancer. Immunohistochemistry may be indicated in prospectively investigating the correlation between expression of topoisomerases and results of chemotherapy treatment. Keywords: DNA topoisomerase; Non-small cell lung cancer; Gene expression; Chemotherapy; Proliferation

1. I n t r o d u c t i o n Abbreviations: SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; topo II-ce, DNA topoisomerase II-c~; topo II-/3, DNA topoisomerase II-/3; topo I, DNA topoisomerase I; ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; SSCP, single strand conformational polymorphism. Corresponding author. Fax: + 31 20 4444355. ~Present address: Netherlands Cancer Institute, Amsterdam, The Netherlands. 2 Present address: Hospital Txagorritxu, Vitoria, Spain. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67-4781(95)00171-9

L u n g cancer is the leading cause of cancer-related death in the W e s t e r n world. T w o major types of lung cancer are clinically recognized: S C L C and a heterogeneous group of histological forms, called N S C L C . A l t h o u g h S C L C is highly sensitive to chemotherapy and radiation, most SCLC patients relapse with a drug refractory disease, and conse-

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quently less than 5% of the patients with SCLC can be cured. The mainstay treatment of NSCLC is surgery and, in contrast to SCLC, NSCLC is only modestly sensitive to chemotherapy, with limited impact on survival [1]. NSCLC and relapsed SCLC constitute prototypes of intrinsic and acquired drug resistance, respectively. Among the mechanisms of drug resistance investigated in lung cancer, the overexpression of MDRI gene is only rarely present in lung cancer cell lines and tumors [2], and thereby does not likely play a major role in determining drug resistance in this malignancy. A reduced expression or activity of topo II is associated with reduced sensitivity to topo II inhibitors, in several cell lines and tumor xenografts, including lung cancer cell lines [3-5]. In addition, mutations of the gene have also been reported to be associated with resistance to topo II inhibitors [4,5]. Reduced topo I activity a n d / o r gene expression, or mutations, have also been suggested to be responsible for the development of drug resistance to topo I inhibitors [4,5]. DNA topoisomerases are ubiquitous enzymes, which change DNA conformation, during important steps of DNA metabolism, such as DNA synthesis, transcription, chromosomal segregation and recombination [6,7], which require uncoiling of DNA. There are two major types of DNA topoisomerases, topo I and II, functionally distinct because topo I cuts one strand of DNA, whereas topo II cuts both strands of DNA at one time, and requires ATP for its function. Recently, two isoforms of topo II, called c~ and /3, of 170 and 180 kDa molecular mass, respectively, have been discovered in mammalian cells [8], which are coded by different genes, localized on chromosome 17q2122 [9] and on chromosome 3p24 [10], respectively. Despite the high degree of homology, the two topo II isoforms are differentially regulated, have distinct properties and probably different functions [8,10-15]. Topo II-c~ and to a lesser extent topo I levels are sensitive to changes in cell proliferation state, being high during rapid proliferation and low when cells have reached a plateau of growth [8], whereas topo 1I-/3 expression is rather stable during different phases of the cell cycle [15]. Drugs commonly used in the treatment of SCLC and NSCLC are potent topo II inhibitors (e.g., anthracyclines and epipodophyllotoxins). It is likely that the two topo II isoforms have different sensitivities to cytotoxic drugs [8]. Moreover, water soluble derivatives of camptothecin, a potent topo I inhibitor, have recently been shown to be active in the treatment of lung cancer [16,17]. DNA topo inhibitors interfere with the religation step of enzyme activity, thus a stable cleavable complex is formed between the enzyme, DNA and the drug, which prevents the resealing of strand breaks. The formation of these DNA strand breaks eventually leads to cell death [18,19]. Although the expression and activity of topo enzymes has been extensively investigated in cell lines, only a few reports described the expression of the topo genes in human tissues and malignancies [20-25], and its implica-

tion with therapy. In this article we describe the expression of DNA topo genes, with particular attention to topo II-a, in normal human lung and NSCLC. The observed differential expression of the topo genes in neoplastic versus normal lung indicates differential function of these genes and probably different sensitivity to anticancer drugs.

2. Materials and methods 2.1. Patient material

Tumor samples obtained from 60 patients who underwent surgery for a primary NSCLC in two different institutions were snap frozen during operation in liquid nitrogen and then preserved at - 8 0 ° C until assayed. For 25 patients, normal lung tissue was also obtained. 70-100 cryosections 20 /~m thick were cut and used for nucleic acid extractions, while the first and last sections were used for histologic assessment (H and E staining). All samples were evaluated for percentage of necrotic tissue, normal infiltrating cells, and tumor cells; only samples in which a substantial part ( > 80%) of the sample was represented by tumor cells were evaluated. 2.2. Nucleic acid extractions, Northern and Southern blotting

Frozen sections were lysed in guanidinium isothiocyanate, and total RNA and DNA were simultaneously extracted by separation and purification in a cesium chloride gradient [26]. Total RNAs extracted from several human lung cancer cell lines, growing in exponential phase [27], were also used. Northern and Southern blotting were performed as previously described [27]. DNA samples were examined in Southern blot experiments after digestion with EcoRI a n d / o r PstI. For Northern and Southern blotting a 1.8 kb topo II-a cDNA fragment (h-TOP2-Z2) [9], kindly provided by Dr. L. Liu, was used. Quantification of gene expression by Northern blotting was performed by densitometry of autoradiographs. Membranes were stripped and rehybridized with GAPDH or /3-actin probes, whose expression served to correct for amount of RNA loading, as previously described [27]. 2.3. RNase protection assay

Because topo II-c~ gene expression as assessed by Northern blotting proved to be insufficiently sensitive, an RNase protection assay was employed. [c~-3Zp]-labeled RNA complementary to topo II-a cDNA sequences (nucleotides 1277-2440) [28], inserted into pGEM4 XbaI site, was transcribed from BglII-linearized DNA, using SP6 RNA polymerase. RNase protection was carried out as described [29]. Protected probe was visualized by elec-

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trophoresis through a denaturing 6% acrylamide gel, followed by autoradiography. In all experiments a probe for y-actin was included as control for amount of RNA loading [29], and human lung cancer cell lines were used for comparison of expression levels between different experiments. The amount of topo mRNA relative to the amount of y-actin mRNA was calculated by densitometric scanning of autoradiographs. Expression of topo II-/3 gene was also assessed by RNase protection assay, employing a specific 702 bp nucleotide sequence CAA5 [30], inserted into the EcoRI site of BluescriptII SK + , linearized by Hinfl restriction and transcribed using T3 polymerase. Expression of topo I was also assessed by RNase protection in a limited number of samples, employing a 703 bp sequence (nucleotides 835-1538)[31], inserted into the EcoRI site of pGEM3, linearized by FokI restriction using T7 polymerase.

2.5. Statistical analysis Linear regression analysis and Pearson test were used to investigate the presence of correlations between the gene expression assessed by different methods [34]. The correlation between gene expression and prognostic factors was investigated by the chi-square test, Gene expression was studied as a continuous variable or taking the median expression level as cut-off point. The influence on survival of the known prognostic factors, and the expression of the tested genes was also evaluated. Survival curves were performed using the Kaplan-Meier method, while comparisons of survival curves were performed by Mantel-Cox analysis [35].

3. Results

3.1. Patient population 2.4. lmmunohistochemistr 3, 4 /zm thick sections from frozen tissues and cytospins of human lung cancer cell lines were used for immunohistochemistry. Slides were dried overnight and fixed at room temperature for 10' in 100% acetone for the antibodies directed against Ki-67, topo I, topo II-/3, or in 4% paraformaldehyde plus 5% acetic acid for topo II-o~. Slides were first preincubated with 2% normal rabbit serum in 1% B S A / P B S and then incubated with the specific antibodies for 1 h at room temperature. Controls were handled simultaneously substituting the specific antibody for mouse IgG. Slides were then incubated with the second rabbit antimouse IgG + A + M biotinylated antibody (Zymed Laboratories Inc., CA, USA) for 30'. Finally slides were incubated with a 1:500 diluted streptavidine-horseradish peroxidase conjugate (Zymed) for 60'. For development of the peroxidase stain, slides were incubated for 3-5' at room temperature with freshly prepared diaminobenzidine (0.5 m g / m l ) and 0.07% hydrogen peroxide in PBS and rinsed with tap water. Slides were briefly counterstained in hematoxylin, rinsed in water and after dehydration mounted with Depex mounting medium (BOH Laboratories supplies, Poole, UK). Mouse monoclonal antibodies for topo II-ce, topo II-/3 and topo I were kindly provided by Dr. G. Astaldi-Ricotti as supernatant of hybridomas [32]. Topo II-c~ antibody 6G2 is a IgG1 K, and was used diluted 1:50. Topo II-/3 antibody 8F8 is a IgG2a K and was used diluted 1:25. Topo I antibody 6B5 is a IgG2a K, and was used diluted 1:25. In addition samples were also stained with the monoclonal antibody Ki-67 (Dako, Glostrup, Denmark), diluted 1:10, in order to evaluate proliferation rate of the tumor [33]. The percentage of tumor cells which stained with each antibody, was estimated by a pathologist (PvdV) and one of us (GG).

Characteristics, history and follow-up were available in all patients (Table 1). Median follow-up was 33 months. No patient received adjuvant chemotherapy or radiotherapy after operation, and the vast majority received only palliative treatment at relapse. 3.2. Expression of DNA topo ll-ee Expression of DNA topo II-o~ was investigated by three methods: Northern blotting, RNase protection, and immunohistochemistry. Northern blotting was found to be a

Table 1 Patient characteristics Total number Median age (range), years Sex: m a l e / f e m a l e Histology squamous adenocarcinoma large cell adenosquamous undifferentiated Surgery pneumonectomy lobectomy exploration (M 1) L y m p h node status (p) NO NI-2 Nx Stage 1

11 Ilia llIb IV

60 64 (44-77) 50/10 29 15 1I 4 I 2I 38 1 3O 29 I 22 22 14 I I

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suboptimal technique, because only 54% (46/85) samples were adequately evaluable, mainly due to insufficient RNA or degradation. None of the normal lung samples expressed detectable topo II-a gene transcripts, while 57% of evaluable NSCLC samples expressed the gene in detectable amounts (not shown). By RNase protection assay, the majority of samples (93%, 79/85) was adequately evaluable, because this technique is less sensitive to RNA degradation than Northern blotting, as it uses a relatively small protected probe [26] (Table 2 and Fig. 1). Interestingly, although two thirds of the normal lung cases did not have appreciable expression, 32% of them had a faint band corresponding to the topo II-a transcript. In all the samples examined by Northern blotting (not shown) or RNase protection (Fig. 1), a normally sized transcript or protected fragment was observed. Most of the tumors had readily detectable levels of gene expression, and expression of tumors was always definitely higher than that of the normal lung counterpart in the same patient's material. The median level of expression was 10% of that of a relatively chemosensitive SCLC cell line, NCI-H1284 [27], used as control in all experiments. In 7 samples however, no expression could be detected. Topo II-a expression levels as assessed by Northern blotting and by RNase protection assay were highly correlated in all samples (n = 43, R = 0.621, P = 0.00001) and in tumor

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Table 2 DNA topo II-oe and topo 1I-/3 gene expression by RNase protection assay

Normal lung evaluable quantifiable undetectable detectable not quantifiable median expression level J (range): NSCLC evaluable quantifiable undetectable detectable not quantifiable median expression level i (range):

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22 0 (0%) 15 (68%) 7 (32%) - (-)

12 12 (100%) 0.13 (0.03-0.24)

57 48 (84%) 7 (12%) 2 (4%) 0.1 (0-0.3)

30 30 (100%) 0.08 (0.02-0.29)

See Section 2 for details; total RNA extracted from normal lung and NSCLC samples was hybridized with specific topo II-o~ and topo II-,8 RNA probes, and a y-actin probe, and run on an acrylamide gel; densitometric scanning of autoradiographs was then performed, and relative topo II gene expression corrected by expression of y-actin. I Relative to gene expression level of NCI-HI284 cell line, arbitrarily set as expression level = I.

samples only (n = 33, R = 0.567, P = 0.00058) (not shown). In a selected number of tumor samples, topo II-o~ expression was evaluated by immunohistochemistry. The

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Fig. 1. RNase protection assay for topo II-o~ gene expression. See Section 2 for details on the technique. A number of NSCLC (T) samples and paired samples of normal lung (N) and NSCLC (T) from the same patient are shown. Histology is also indicated (SQ = squamous, AD = adenocarcinoma. LC = large cell, AS = adenosquamous). Expression of topo II-a is always higher in the tumor than in normal lung, where it is mostly undetectable. NCI-HI284 is a human SCLC cell line, used as positive control, and t-RNA was used as negative control. Correction for loading of RNA was performed in the same experiment by using a y-actin probe as indication of RNA loading, and quantifying the relative topo II-a expression by densitometric scanning of autoradiographs. Molecular weights of the transcribed probes before RNase treatment, and of the protected fragments are shown.

G. Giaccone et al. / Biochimica et Biophysica Acta 1264 (1995) 337-346

staining pattern was mainly nuclear, but in some instances a specific cytoplasmatic staining was also present (Fig. 2). Staining was almost exclusively detected in tumor cells. Most tumor samples had detectable topo II-~ protein expression (n = 31; median number of positive cells 70%, range 0 - 1 0 0 % ) . In only one tumor sample no expression was detectable by immunohistochemistry, which correlated with absence of detectable expression by RNase protection experiments. In tumor samples there was a good correlation between topo I I - a gene expression as determined by RNase expression, and by immunohistochemistry (n = 30, R = 0.49, P = 0.0059) (not shown).

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3.3. Structure analysis o f the topo ll-ce gene by Southern blotting

Because in several in vitro systems gene rearrangements and mutations of the topo II-c~ gene have been reported to be responsible for reduced sensitivity to topo II-targeted anticancer agents [3-5], we investigated whether gross gene rearrangements of this gene were present in NSCLC. Genomic DNAs extracted from 50 NSCLC samples were analyzed by Southern blotting. No difference in restriction patterns were identified with 1 ( n = 8 ) or 2 ( n = 4 2 ) restriction enzymes, and no amplification of the gene was

Fig. 2. Immunohistochemistry using specific antibodies for topo I, topo II-a, and topo lI-fl, of a squamous cell lung cancer ( × 1000). See Section 2 for details. Panel A, control; panel B, staining with topo I antibody, the staining is granular nuclear + nucleolar; panel C, staining with topo I1-o~antibody, the staining is diffuse nuclear excluding the nucleolus (arrow), and localized in tumor cells (t) but not in stromal cells (s); panel D, staining with topo 1I-/3 antibody, the staining is exclusively nucleolar, and involves also stromal cells (arrow).

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observed, indicating that in untreated NSCLC gross abnormalities in the topo II-a gene are not common (not shown).

3.4. Expression of DNA topo ll-fi By RNase protection assay, topo I1-/3 expression was clearly detectable in all the normal lung and NSCLC samples tested, in contrast to topo II-a expression (Table 2 and Fig. 3). The relative level of topo II-/3 gene expression in tumors was slightly lower than that of topo II-ce and the range of expression levels was narrower. Interestingly, although the level of expression of normal lung and NSCLC varied within couples of normal/tumor samples, and the ratio normal lung/tumor in 10 paired samples was 1.2 (range 0.3-8.9), there was no indication of a preferential expression of topo II-/3 in normal lung or in tumors. By immunohistochemistry (n -- 33), a characteristic nucleolar staining, was observed in virtually all tumor samples examined, as previously described [36], and staining was present also in normal lung tissue cells and in stromal cells. The percentage of tumor cells stained with the antibody ranged from 0% (2 samples) to 100% (median 80%).

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There was no correlation between expression of topo 11-/3 obtained by RNase protection and by immunohistochemistry (not shown), probably because of the confounding effect of the high expression in normal lung tissue and normal infiltrating cells. There was no correlation between expression of topo II-a and -/3 genes obtained by any technique (not shown).

3.5. Expression of DNA topo I By immunohistochemistry (n = 33), typical granular staining was localized in the nucleus and nucleoli (Fig. 2), and tumor cells were stained definitely more frequently than the surrounding normal lung parenchyma and stroma. However, also within normal tissue were there occasional positively stained areas. There was a wide variation in the number of cells expressing the antigen, ranging from 0% ( 9 / 3 3 = 27%) to 100% (median 40%). No correlation was found between expression of topo I and that of the two topo II genes (not shown). Expression of topo I was also assessed by RNase protection in a limited number of samples, which confirmed the lower expression of this gene in normal lung as compared to the tumors. Interestingly, in contrast to ex-

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Fig. 3. RNase protection assay for topo 1I-,8 gene expression. See Section 2 for details on the technique. A number of NSCLC (T) samples and paired samples of normal lung (N) and NSCLC (T) from the same patient are shown. Histology is also indicated (SQ = squamous, AD = adenocarcinoma, AS = adenosquamous). Expression of topo 11-/3 is similar in the tumor and in the normal lung. NCI-HI284 and NCI-N417 are human SCLC cell lines used as positive control, and t-RNA was used as negative control. Correction for loading of RNA was performed in the same experiment by using a y-actin probe as indication of RNA loading, and quantifying the relative topo 11-/3 expression by densitometric scanning of autoradiographs. Molecular weights of the transcribed probes before RNase treatment, and of the protected fragments are shown.

G. Giaccone et al./Biochimica et Biophysica Acta 1264 (1995) 337-346

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Fig. 4. RNase protection assay for topo I gene expression. See Section 2 for details on the technique. Some NSCLC (T) samples and paired samples of normal lung (N) and NSCLC (T) from the same patient are shown. Histology is indicated (SQ = squamous, AD = adenocarcinoma, LC = large cell, AS = adenosquamous). Expression of topo I is higher in the tumor than in the normal lung. NCI-N417, NCI-HI87, NCI-H209, NCI-H1284, and NCI-H69 are human SCLC cell lines; NCI-H322, NCI-H1355, and NCI-H23 are NSCLC cell lines. Correction for loading of RNA was performed in the same experiment by using a y-actin probe as indication of RNA loading, and quantifying the relative topo I expression by densitometric scanning of autoradiographs. Molecular weights of the transcribed probes before RNase treatment, and of the protected fragments are shown.

pression of topo II genes, topo I levels of expression in NSCLC samples were in the range of those observed in several human lung cancer cell lines (Fig. 4).

was present in a median of 10% of tumor cells (n = 33, range 0 - 8 0 % ) , and the association between expression of topo II-c~, as measured by immunohistochemistry, and expression of Ki-67 was of borderline statistical significance (n = 29, R = 0.353, P = 0.060) (Fig. 5A). In contrast, no association was found between Ki-67 and topo II-/3 (Fig. 5B) or topo I (Fig. 5C).

3.6. Expression of the proliferation marker Ki-67 The expression of the proliferation marker Ki-67 was also investigated in tumor samples. Expression of Ki-67

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Fig. 5. Correlation graphs between percentage of cells staining positive by immunohistochemistry for expression of Ki-67 and topo ll-ot (n = 29, R = 0.353, P = 0.060) (panel A), Ki-67 and topo II-fl (n = 31, R = 0.045, P = NS) (panel B), and Ki-67 and topo I (n = 31, R = 0.08, P = NS) (panel

C).

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3.7. Relationship between clinical features, survival and expression o f DNA topoisomerases

No significant difference in expression of any of the topo genes was found for different sex, age, tumor histology, grading, lymph node status and stage. Similarly, no significant difference in expression of Ki-67 was found for different sex, age, lymph node status and stage. However, a significantly higher Ki-67 score was observed in large cell and undifferentiated carcinomas (43%) than in squamous and adenocarcinomas (15%) (n = 27 vs. 6; P = 0.01), and there was a trend for poorly differentiated carcinomas (grade 3) to express higher Ki-67 than moderately and well differentiated tumors (14% vs. 23%, respectively; P = 0.09). The median survival time of the whole population was 22 months. Sex and tumor grading did not affect survival, whereas stage ( P = 0.008), lymph node status ( P = 0.036), and histology (squamous vs. non-squamous, P = 0.044) had a significant influence on survival. By taking the median levels of expression of the topo genes and Ki-67 as the cut-off values, there was no significant difference in survival of high versus low expression of any of the genes or gene products tested. Although not significant, a trend was found for a higher expression of topo II-a, by RNase protection ( > 0 . 1 units, relative to NCI-H1284 cells) to have a negative impact on survival than lower expression levels ( < 0.1, relative to NCI-H1284 cells) (median survival time 19 vs. 26 months, respectively; P = 0.3). Moreover, the median survival of patients with < 10% of Ki-67 positively stained cells was longer than that of patients with > 10% positive cells (12 vs. 21 months, respectively; P = 0.4).

4. Discussion 4.1. Differential expression o f DNA topo H genes

The median level of mRNA expression of the two topo II isoforms in NSCLC was approximately one tenth of that of a relatively chemosensitive SCLC cell line. Although virtually absent in normal lung parenchyma and stromal cells, topo II-oz was expressed in most tumors. In contrast, similar levels of topo II-/3 were expressed in normal lung and NSCLC. The cellular localization of the two topo II isozymes was strikingly different, being the a form localized mainly diffusely in the nucleus, and the /3 form being restricted to the nucleoli. All together, these results suggest a different function of the two topo II genes in the lung. Zini et al. [36] showed that the p180 enzyme is localized mainly in the dense fibrillar component of the nucleolus; in contrast, topo I was present in both nucleoplasm and nucleolar region by E.M. immunohistochemistry, and topo II-o~ was detected only in the nucleoplasm and mostly spared the nucleolus [37]. Given the distinct localization of

the two topo II enzymes in the nucleus, the c~ form has been suggested to be involved in DNA synthesis, and the /3 form in transcription [36]. As in our study, no obvious relationship in terms of level of expression between topo I I - a and II-/3 genes has been detected in a number of cell lines [38], again indicating a differential gene regulation. However, in AML, a strong correlation of expression of the two topo II isoforms has been shown [20], whereas no correlation was found in a number of solid tumors [21], indicating possible differences between tumor types. The two topo II isoforms have different patterns of expression in normal murine tissues, in fact the /3 form is also expressed in non-proliferating tissues, suggesting a role in DNA processes other than replication [39,40]. Expression of topo II-o~ appears to be lower in tumor tissues than in cell lines and this is to a great extent dependent on the higher proliferative state of cell lines, compared to original tumors. However, we found lower expression of both topo II genes in NSCLC than in human lung cancer cell lines. Similar findings were obtained in AML samples, as compared to HL-60 cells [20]. A correlation between activity/expression of topo II, drug-mediated DNA cleavage and sensitivity to topo II poisons has been reported in several drug resistant cultured cell lines, selected by prolonged exposure to topo II inhibitors, as well as in unselected cell lines [3-5]. Higher levels of expression and activity of topo II have been observed in SCLC than in NSCLC cell lines, which might partially explain the higher response of SCLC to etoposide and doxorubicin [41]. A relative resistance during quiescence phases has been shown for several cell lines, human lymphocytes and murine tissues [42-44], and this is in agreement with the low topo II-o~ expression in slowly proliferating cells [45,46]. Several mechanisms have been suggested to be responsible for the reduced expression, i.e., gene hypermethylation [47], post-translational modifications of the protein [46], and altered phospborylation [48]. Although mutations have been identified mainly in in vitro drug resistant selected cell lines and only a few unselected cell lines [3-5], no mutations have so far been described in human malignancies. In our study, we could not identify any gross rearrangement or amplification of the topo II-c~ gene by Southern blotting. Because point mutations in functionally important areas of the gene have been described in cell lines, Southern blotting may not be sufficiently sensitive for screening. Nevertheless, in a study of 15 relapsed ALL patients [49], and in samples from 23 patients with untreated AML [20], no mutations could be identified even by PCR-based SSCP analysis. It is still unclear which topo II isoform is more important for the interaction with cytotoxic drugs. Topo II-/3 is 3- to 4-fold less sensitive to inhibition by intercalating agents and epipodophyllotoxins than the o~ form [8], and in general its expression is not reduced in resistant cells.

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However, in HL-60 cell line resistant to mitoxantrone [50] and in Susa testicular cell lines with moderate degree of resistance to etoposide [51], reduced topo II-o~ expression and very low expression of topo II-/3 were observed. This might indicate that also topo II-/3 could in part account for drug resistance to known topo II inhibitors. Of interest in our study is that whereas relatively high topo II-a expression was only expressed in tumor tissue, high topo II-/3 expression was also seen in normal tissue. It is therefore unlikely that topo II-/3 be the major target of topo II inhibitors, at least in lung, as no lung toxicity is induced by topo II inhibitors.

4.2. Expression of DNA topo I gene We found a low level of expression of topo I in normal lung by immunohistochemistry, in contrast to relatively high levels in three fourths of the NSCLC samples. The pattern of nuclear distribution was different from that of topo II-a, as in fact it includes nucleolar regions, which were spared by the latter. By RNase protection assay, the relative expression of normal lung and NSCLC was confirmed and, interestingly, the levels of expression in NSCLC samples were comparable to those of human lung cancer cell lines. This contrasts with the definitively lower levels found for both topo II genes. Topo 1 expression was higher in colon [22,52], and prostate tumors than normal counterparts, but no difference was found in kidney tumors versus normal kidney, indicating that topo I expression is tumor type specific [22]. The results of expression of topo I in NSCLC and colon cancer would indicate an advantage in using topo I inhibitors in these malignancies. Strikingly, CPT-I 1, a novel camptothecin derivative, has recently shown remarkable antitumor activity in advanced NSCLC [16] and in colorectal cancer refractory to 5-FU containing chemotherapy

[53]. 4.3. Proliferation. expression (~[ the topo genes and prognosis In our study, higher expression levels of topo II-c~ were in general associated with higher expression of Ki-67. In contrast, no correlation was present between expressions of Ki-67 and topo II-j3 or topo I. A similar correlation between topo II-oz expression and Ki-67 has been shown in a number of solid tumors [21]. The expression levels of any of the topo genes was not correlated to any of the characteristics of the patients or their tumors. Ki-67 staining has been used to determine the growth fraction in a number of tumors, e.g., lymphomas, lung, brain and breast carcinomas. It has been shown to be predictive of shorter progression free survival [54] or survival [55] in radically resected NSCLC. In our study, there was a trend towards a poorer survival in patients with more than 10% cells positive for Ki-67 staining, although

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it did not reach a significant level, probably due to the relatively small number of patients. Interestingly, although not significant, a 7 month longer median survival was observed in relatively low as compared to high levels of topo II-c~ gene expression, suggesting its possible prognostic role in NSCLC.

4.4. Topo H expression and response to chemotherapy Reduced levels of topo genes appear to be the most common mechanism of cellular resistance to topo inhibitors in vitro, and the quantification of expression a n d / o r activity of topoisomerases would be desirable to identify patients' sensitivity to treatment. Interestingly, lower levels of topo II expression were present in ovarian cancer patients resistant to a topo II unrelated chemotherapy (cisplatin-cyclophosphamide) [23]. Topo II-c~ was expressed in 65% of 54 ovarian tumors, at higher levels in stage IV and grade llI [24]. In contrast, topo II-/3 and I were expressed in all ovarian tumors examined [24]. In a recent study, no correlation was found between topo II expression and resistance to doxorubicin as determined by a short term in vitro cytotoxicity test, in 48 surgically resected squamous cell carcinomas of the lung [25]. However, in this study, immunohistochemistry was performed using a polyclonal antibody which does not distinguish between the two topo II isoforms. In AML patients [20] no correlation was found between topo II-c~ expression, as assessed by Western blotting, and patient response to induction chemotherapy. Similarly, in childhood ALL no correlation was found between topo II-~ expression, as assessed by RNase protection assay, and in vitro sensitivity to topo inhibitors of fi'esh leukemic cells, and no difference in levels of expression were seen between newly diagnosed patients and relapsing patients [56]. In conclusion, differential expression and different subcellular distribution of the topo genes was found in normal lung and NSCLC. While topo II-c~ and I are expressed primarily in tumor tissues, topo II-/3 is expressed also in normal tissue at relatively high levels. This suggests different functions of the two topo II genes and different regulation in normal lung and lung tumor tissue, and should stimulate the search for new potent and specific topo II-c~ inhibitors and topo I inhibitors. The prospective investigation of expression a n d / o r activity of topo genes in patients' material, as a tool to identify chemoresistance is indicated.

References [1] Ginsberg, R.J., Kris, M.G. and Armstrong, J.G. (1993) In Cancer Principles and Practice of Oncology (DeVita, V.T., Hellman, S. and Rosenberg, S.A., eds.), 3rd Edn., pp. 673-758, Lippincott, Philadelphia.

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[2] Lai, S.L., Goldstein, L.J., Gottesman, M.M., Pastan, 1., Tsai, C.M., Johnson, B.E., Mulshine, J.L., Ihde, D.C., Kayser, K. and Gazdar, A.F. (1989) J. Natl. Cancer Inst. 81, 1144-1150. [3] Hochhauser, D. and Harris, A.L. (1993) Cancer Treat. Rev. 19, 181-194. [4] Giaccone, G. (1994) Pathol. Biol. 4, 346-352. [5] D'Incalci, M., Capranico, G., Giaccone, G., Zunino, F. and Garattini, S. (1993) In Cancer chemotherapy and biological response modifiers (Pinedo, H.M., Longo, D.L. and Chabner, B.A., eds.), annual 14, pp. 61-85. [6] Wang, J.C. (1987) Biochim. Biophys. Acta 909, 1-9. [7] Osheroff, N. (1989) Pharm. Ther. 41,223-241. [8] Drake, P.H., Hofmann, G.A., Bartus, H.F., Mattern, M.R., Crooke, S.T. and Mirabelli, C.K. (1989) Biochemistry 28, 8154-8160. [9] Tsai-Pflugfelder, M., Liu, L,F., Liu, A.A., Tewey, K.M., Whan-Peng, J., Knutsen, T., Huebner, K., Croce, C.M. and Wang, J.C. (1988) Proc. Natl. Acad. Sci. USA 85, 7177-7181. [10] Tan, K.B., Dorman, T.E., Falls, K.M., Chung, T.D.Y., Mirabelli, C.K., Crooke, S.T. and Mao, J. (1992) Cancer Res. 52, 231-234. [11] Chung, T.D., Drake, F.H., Tan, K.B., Per, S.R., Crooke, S.T. and Mirabelli, C.K. (1989) Proc. Natl. Acad. Sci. USA 86, 9431-9435. [12] Hsiang, Y.H., Wu, H.Y. and Liu, L.F. (1988) Cancer Res. 48, 3230-3235. [13] Zwelling, L.A., Hinds, M., Chan, D., Altschuler, E., Mayes, J. and Zipf, T.F. (1990) Cancer Res. 50, 7116-7122. [14] Woessner, R.D., Chung, T.D.Y., Hofmann, G.A., Mattern, M.R., Mirabelli, C.K., Drake, F.H. and Johnson, R.K. (1990) Cancer Res. 50, 2901-2908. [15] Woessner, R.D., Mattern, M.R., Mirabelli, C.K., Johnson, R.K. and Drake, F.H. (1991) Cell. Growth Differ. 2, 209-214. [16] Fukuoka, M., Nitani, H., Suzuki, A., Motomiya, M., Hasegawa, K., Nishikawi, Y., Kuriyama, T., Ariyashi, Y., Negoro, S., Masuda, N., Nakajima, S. and Taguchi, T. (1992) J. Clin. Oncol. 10, 16-20. [17] Masuda, N., Fukuoka, M., Kusunoki, Y., Matsui, K., Takifuji, N., Kudoh, S., Negoro, S., Nishioka, M., Nakagawa, K. and Tokada, M. (1992) J. Clin. Oncol. 10, 1225-1229. [18] Liu, L.F. (1989) Annu. Rev. Biochem. 58, 351-375. [19] D'Arpa, P. and Liu, L.F. (1989) Biochim. Biophys. Acta 989, 163-177. [20] Kaufmann, S.H., Karp, J.E., Jones, R.J., Miller, C.B., Schneider, E., Zwelling, L.A., Cowan, K., Wendel, K. and Burke, P.J. (1994) Blood 83, 517-530. [21] D'Andrea, M.R., Farber, P.A. and Foglesong, P.D. (1994) Appl. Immunhistol. 2, 177-185. [22] Husain, I., Mohler, J.L., Seigler, H.F. and Besterman, J.M. (1994) Cancer Res. 54, 539-546. [23] Van der Zee, A.G.J., Hollema, H., De Jong, S., Boonstra, H., Gouw, A., Willemse, P.H.B., Zijlstra, J.G. and De Vries, E.G.E. (1991) Cancer Res. 51, 5915-5920. [24] Van der Zee, A.G.J., De Vries, E.G.E., Hollema, H., Kaye, S.B., Brown, R. and Keith, W.N. (1994) Ann. Oncol. 5, 75-81. [25] Volm, M., and Mattern, J. (1992) Carcinogenesis 13, 1947-1950. [26] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. [27] Giaccone, G., Gazdar, A.F., Beck, H., Zunino, F. and Capranico, G. (1992) Cancer Res. 52, 1666-1674. [28] Binaschi, M., Giaccone, G., Supino, R., De Isabella, P., Astaldi, G., Gazdar, A.F., Capranico, G. and Zunino, F. (1992) J. Natl. Cancer Inst. 84, 1710-1716. [29] Baas, F., Jongsma, A.P.M., Broxterman, H.J., Arceci, R.J., Housman, D., Scheffer, G.L., Riethorst, A., Van Groeningen, M., Nieuwint, W.M. and Joenie, H. (1990) Cancer Res. 50, 5392-5398.

[30] Austin, C.A. and Fisher, M. (1990)FEBS Lett. 266, 115-117. [31] Juan, C.C., Hwang, J., Liu, A.A., Whang-Peng, J. Knutsen, T., Huebner, K., Croce, C.M., Zhang, H., Wang J.C. and Liu, L.F. (1988) Proc. Natl. Acad. Sci. USA 85, 8910-8913. [32] Negri, C., Chiesa, R., Cerino, A., Bestagno, M., Sala, C., Zini, M., Maraldi, N.M. and Astaldi-Ricotti, G.C.B. (1992) Exp. Cell Res. 200, 452-459. [33] Gerdes, J., Schwab. U., Lemke, H. and Stein, H. (1983) Int. J. Cancer 31, 13-20. [34] Armitage, P. and Berry, G. (1978) Statistical methods in medical research, Blackwell, Oxford. [35] Dixon W.J. (1990) DMDP Statistical Software Manual, University of California Press, Berkeley, [36] Zini, N., Martelli, A.M., Sabatelli, P., Santi, S., Negri, C., AstaldiRicotti, G.C.B. and Maraldi, N.M. (1992) Exp. Cell Res. 200, 460-466. [37] Zini, N., Santi, S., Ognibene, A., Bavelloni, A., Neri, L.M.. Valmori, A., Mariani, E., Negri, C., Astaldi-Ricotti, G.C.B. and Maraldi, N.M. (1994) Exp. Cell Res. 210, 336-348. [38] Jenkins, J.R., Ayton, P., Jones, T., Davies, S.L., Simmons, D.L., Harris, A.L., Sheer, D. and Hickson, I.D. (1992) Nucleic Acids Res. 20, 5587-5592. [39] Capranico, G., Tinelli, S., Austin, C.A., Fisher, M.L. and Zunino, F. (1992) Biochim. Biophys. Acta 1132, 43-48. [40] Juenke, J.E.M. and Holden, J.A. (1993) Biochim. Biophys. Acta 1216, 191-196. [41] Kasahara, K., Fijiwara, Y., Sugimoto, Y., Nishio, K., Tamura, T., Matsuda, T. and Sanijo, N. (1992) J. Natl. Cancer Inst. 84, 113-118. [42] Zwelling, L.A., Estey, E., Silberman, L., Doyle, S. and Hittelman. W. (1987) Cancer Res. 47, 251-257. [43] Heck, M.M.S. and Earnshaw, W.C. (1986) J. Cell. Biol. 103, 2569-2581. [44] Markovits, J., Pommier, Y., Kerrigan, D., Covey, J.M., Tilchen, E.J. and Kohn, K.W. (1987) Cancer Res. 47, 2050-2055. [45] Hwang, J., Shyy, S., Chen, A.Y., Juan, C. and Whang-Peng, J. (1989) Cancer Res. 49, 958-962. [46] Heck, M.M.S., Hittelman, W.N. and Earnshaw, W.C. (1988) Proc. Natl. Acad. Sci. USA 85, 1086-1090. [47] Tan, K.B., Mattern, M.R., Eng, W.K., McCabe, F.L. and Johnson, R.K. (1989) J. Natl. Cancer Inst. 81, 1732-1735. [48] Takano, H., Kohno. K., Ono, M., Uchida, Y. and Kuwano, M. (1991) Cancer. Res. 51, 3951-3957. [49] Danks, M.K., Warmoth, M.R., Friche, E., Granzen, B., Bugg, B.Y., Harker, W.G., Zwelling, L.A., Futcher, B.W., Suttle, D.P. and Beck, W.T. (1993) Cancer Res. 53, 1373-1379. [50] Harker, W.G., Slade, D.L., Drake, F.H. and Parr, R.L. (1991) Biochemistry 30, 9953-9961. [51] Hosking, L.K., Whelan, R.D.H., Shellard, S.A., Davies, S.L., Hickson, I.D., Danks, M.K. and Hill, B.T. (1994) Int. J. Cancer 57, 259-267. [52] Giovanella, B.C., Stehlin, J.S., Wall, M.E., Wani, M.C., Nicholas, A.W., Liu, L.F., Silber, R. and Potmesil, M. (1989) Science (Washington) 246, 1046-1048. [53] Shimada, Y., Yoshino, M., Wakui, A., Nakao, I., Futatsuki, K., Sakata, Y., Kambe, M., Taguchi, T. and Ogawa, N. (1993) J. Clin. Oncol. I 1, 909-913. [54] Scagliotti, G.V., Micela, M., Gubetta, L., Leonardo, E., Cappia, S., Borasio, P. and Pozzi, P. (1993) Eur. J. Cancer 29A, 363-365. [55] Tungekar, M.F., Gatter, K.C., Dunnill, M.S. and Mason, D.Y. (1991) Histopathology 19, 545-550. [56] Klumper, E., Giaccone, G., Pieters, R., van Ark-Otte, J., Huismans, D.R., Loonen, A.H., Broekema, G.J., van Wering, E.R., Kaspers, G.J.L. and Veerman, A.J.P. (1995) Leukemia (in press).