Takashi Niimi

Determinants of sensitivity and resistance to gemcitabine: The roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non‐small cell lung cancer

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT‐PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non‐small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r=‐0.6769, P=0.0005), whereas dCK expression levels were not. In a highly gemcitabine‐sensitive cell line, NCI‐H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI‐H23 cells to gemcitabine and subsequently established the drug‐resistant clone H23/GEM‐R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.

The determinants of sensitivity and acquired resistance to gemcitabine differ in non-small cell lung cancer: a role of ABCC5 in gemcitabine sensitivity

We examined the expression levels of the multidrug resistance protein 5 (ABCC5) gene in non–small cell lung cancer (NSCLC) cell lines to clarify the relationship with the sensitivity to gemcitabine. The expression levels of ABCC5 were inversely correlated with gemcitabine sensitivity significantly (r = 0.628; P < 0.01) in 17 NSCLC cells, whereas the expression of ABCC5 in the gemcitabine-resistant NSCLC cell line H23/GEM-R was the same as that in parental NCI-H23 cells. Treatment with the ABCC5 inhibitor zaprinast altered the sensitivity to gemcitabine in ABCC5-expressing NSCLC cells. In addition, decreasing the expression of ABCC5 by small interfering RNA altered the cytotoxicity to gemcitabine. These results indicate that modulation of ABCC5 activity could be used to increase the gemcitabine sensitivity in NSCLC. Previously, we found a decreased expression of deoxycytidine kinase in H23/GEM-R cells, and further investigation in this study showed an increased expression of ribonucleotide reductase subunit 1 in H23/GEM-R cells. We therefore also examined the effect of modifying the expression of both genes on gemcitabine resistance. We found that using small interfering RNA to decrease the expression of ribonucleotide reductase subunit 1 resulted in a decreased resistance to gemcitabine in H23/GEM-R cells. Furthermore, pretreatment with pemetrexed resulted in an increased deoxycytidine kinase expression concomitant with the alteration of the resistance to gemcitabine in H23/GEM-R cells. The determinants for sensitivity and the acquired resistance in gemcitabine are quite different; nonetheless, modification of these factors may increase the efficacy of gemcitabine in the treatment of NSCLC. [Mol Cancer Ther 2006;5(7):1800–6]

Serum Lysozyme Levels and Clinical Features of Sarcoidosis

Abstract. Serum lysozyme is used as a marker of sarcoidosis disease activity. In this study we examined the association between lysozyme levels and the clinical features of sarcoidosis and thus the clinical usability of this parameter in a large population. One hundred ten sarcoidosis patients from central Japan were examined for clinical features and serum lysozyme level at the first visit to our hospital and on a regular basis thereafter. The sensitivity of lysozyme for predicting sarcoidosis was 79.1%, whereas that of serum angiotensin-converting enzyme (ACE) was 59.0%. Even in the cases without an elevated serum ACE level, a value of 72.1% was obtained. The serum lysozyme level demonstrated a significant tendency to increase with the number of organs involved (p < 0.01). There were significant differences among the four radiographic stages (p < 0.05). The maximum serum lysozyme levels of patients without a disappearance of abnormal shadows on chest radiography within 5 years were significantly greater than those of individuals with a disappearance (p < 0.05). A positive correlation between serum lysozyme and serum ACE levels was observed. Because serum lysozyme is much less specific for sarcoidosis than serum ACE, its diagnostic value may be limited. However, the sensitivity was high even when serum ACE levels were within normal limits and correlated well with clinical features in sarcoidosis. Therefore, this parameter seems suitable for disease monitoring in proven cases.

Role of ABCG2 as a biomarker for predicting resistance to CPT-11/SN-38 in lung cancer

To examine the mechanism of resistance to 7‐ethyl‐10‐hydroxycamptothecin (SN‐38) in lung cancer, we continuously exposed the non‐small‐cell lung cancer (NSCLC) cell line NCI‐H23 to SN‐38 and selected the SN‐38‐resistant clone H23/SN‐38. After 2 months of culturing in SN‐38‐free conditions, H23/SN‐38 cells recovered their sensitivity to SN‐38 and were subsequently established as the revertant H23/SN‐38REV cell line. Because H23/SN‐38 cells show cross resistance to certain anticancer drugs, such as topotecan, etoposide, doxorubicin and mitoxantrone, we examined the gene and protein expression levels of drug efflux transporters of the ATP‐binding cassette (ABC) family. We found that both gene and protein expression of ABCG2/BCRP (ABCG2) in H23/SN‐38 cells was increased compared with that in NCI‐H23 cells and H23/SN‐38REV cells. The cellular accumulation of topotecan in H23/SN‐38 cells was decreased compared with that in NCI‐H23 and H23/SN‐38REV cells, and treatment with reserpine (an inhibitor of ABCG2) increased the cellular accumulation of topotecan in H23/SN‐38 cells. Furthermore, treatment with reserpine also altered the sensitivity of H23/SN‐38 cells to SN‐38. These results indicate that the upregulation of ABCG2 was functional, and related to the resistance of H23/SN‐38 cells to SN‐38. Moreover, we found that gene expression levels of ABCG2 were significantly correlated with the concentration of SN‐38 for 50% cell survival in 13 NSCLC cells (r = 0.592, P < 0.05). The present results indicate that the induction of ABCG2 by SN‐38 does confer acquired resistance to CPT‐11/SN‐38, but the induction of ABCG2 and subsequent drug resistance are reversible. However, the expression level of ABCG2 may be a useful indicator of CPT‐11/SN‐38 activity in lung cancer. (Cancer Sci 2006; 97: 192–198)

Efficacy of docetaxel as a second-line chemotherapy for thymic carcinoma.

Thymic carcinoma is a rare and aggressive tumor, and the efficacy of second-line chemotherapy is still unclear. Here, we reported a case of thymic carcinoma that responded well to the administration of docetaxel alone as a second-line chemotherapy. A 64-year-old woman was diagnosed with thymic carcinoma (squamous cell type) with bone metastasis, and she, therefore, received nedaplatin combined with etoposide and ifosfamide. She responded partially, after which she received irradiation for bone metastasis. Two months after chemotherapy, the thymic carcinoma exhibited gradual regrowth and she experienced shoulder pain. We treated this with docetaxel alone (60 mg/m2 every 4 weeks). After three courses of docetaxel, we observed a partial response and her shoulder pain disappeared. This case demonstrated that docetaxel is effective as a second-line chemotherapy for thymic carcinoma.

Genetic polymorphism of the angiotensin-converting enzyme (ACE) in asthmatic patients.

Angiotensin-converting enzyme (ACE) inactivates bradykinin, substance P and neurokinin A, which are believed to play important roles in the pathogenesis of asthma, especially in neurogenic inflammation. It has recently been shown that an insertion (I)/deletion (D) polymorphism in the ACE gene accounts for variation in serum ACE levels. There are thus three genotypes (insertion homozygote, II; deletion homozygote, DD; heterozygotes, DI). The serum ACE level with the DD type is reported to be about double that of the II type and intermediate in the DI case. In the present study, we examined whether asthma is linked with this ACE gene polymorphism. Seventy-one patients with asthma (27 males and 44 females) and 142 sex- and age-matched healthy controls were determined for their genotype by the polymerase chain reaction (PCR) method. Twenty-five asthmatics demonstrated the II type (35.2%), 37 the DI type (52.1%), and nine the DD type (12.7%). There were no significant differences in the distributions of genotypes and serum ACE levels between healthy controls and patients. No significant differences were evident in serum IgE levels, age at onset, proportion of atopic type patients and severity of asthma among the three genotypes. We did not find any association between asthma and the ACE gene polymorphism in this study.

Differing distributions of CXCR3- and CCR4-positive cells among types of interstitial pneumonia associated with collagen vascular diseases

Interstitial pneumonia (IP) is an important complication in collagen vascular diseases (CVDs). We examined the distribution of helper T cell subsets in lung biopsies of cases of IP associated with CVD (CVD-IP). The tissues from 27 CVD-IP patients with rheumatoid arthritis (RA), 8 with polymyositis or dermatomyositis (PM/DM), and 8 with systemic sclerosis (SSc) were compared with those from 10 patients with idiopathic pulmonary fibrosis (IPF) in our previous study. The expressions of CXCR3 and CCR4 (chemokine receptors associated in vitro with Th1 and Th2 cells, respectively) in the mononuclear infiltrate were analyzed immunohistochemically. The positive cells were semiquantified in fibrosing areas of the CVD-IP and IPF cases. The number of CXCR3-positive cells was significantly greater in RA-IP than in PM/DM-IP, SSc-IP, or IPF, whereas there were fewer CCR4-positive cells in RA-IP, PM/DM-IP, and SSc-IP than in IPF. The CXCR3-/CCR4-positive cells ratio was significantly higher in RA-IP and PM/DM-IP (but not in SSc-IP) than in IPF. These results support previous reports of the dominance of Th2 cells in some SSc-IP and IPF cases. However, Th1-type immune responses may predominate in RA-IP and PM/DM-IP. Our findings suggest that the pathogenesis of CVD-IPs differs with the helper T cell subset.

Analysis of β-tubulin gene alteration in human lung cancer cell lines

We examined lung cancer cell lines for the presence of β-tubulin gene alteration based on a previous report of a relationship between frequent β-tubulin gene mutation in non-small-cell lung cancer and clinical response to taxanes as well as the prognosis. The mutation was defined by analyzing genomic DNA from 31 lung cancer cell lines by direct genomic sequencing using specific primers for the β-tubulin class I gene. We detected only three genetic alterations at nonsplice sites in two introns, and a silent genetic alteration at codon 217 in exon 4. The mutation of the β-tubulin gene was rare; moreover, these genetic alterations were predicted to evoke no biological alteration of the cancer cells. Our data suggest that the β-tubulin gene mutation does not play a major role in the genetic mechanism of resistance to taxanes. In addition, the presence of a closely related family of β-tubulins or pseudogenes was thought to hinder accurate evaluation of the β-tubulin gene.

p185HER-2/neu and p21CIP1/WAF1 Expression in Primary Tumors and Lymph Node Metastases in Non-small Cell Lung Cancer

p185HER‐2/neu, a tyrosine kinase receptor, is one of the target molecules for cancer therapy, and its expression may reduce the sensitivity of tumor cells to anti‐cancer drugs. p21CIP1/WAF1 is a cyclindependent kinase inhibitor, and its expression may also be involved in chemoresistance. Non‐small cell lung cancer (NSCLC) is a potentially systemic disease, and systemic therapies play an important role in its treatment. However, there have been no studies comparing the expression of these molecules between primary and metastatic tumors. We investigated the expression of p185HER‐2/neu and p21CIP1/WAF1 in 57 paired samples of primary NSCLC tumors and corresponding lymph node metastases by immunohistochemistry. Expression of each of p185HER‐2/neu and p21CIP1/WAF1 was highly correlated between primary tumors and lymph node metastases, and similar correlations were also obtained when adenocarcinoma and squamous cell carcinoma cases were analyzed individually. However we failed to detect any correlation between p185HER‐2/neu and p21CIP1/WAF1 expression. Our results suggested that expression of both p185HER‐2/neu and p21CIP1/WAF1 is concordant between primary and metastatic tumors.

Detection of API2-MALT1 Fusion Transcripts in Cytologic Specimens of Patients with Pulmonary Mucosa-Associated Lymphoid Tissue Lymphoma

Mucosa-associated lymphoid tissue (MALT) lymphoma constitutes up to 90% of primary pulmonary lymphomas, but its diagnosis is often difficult.API2-MALT1 fusion is specific to MALT lymphoma and is detected in nearly half of the pulmonary cases. Cytologic examinations have played a pivotal role in the diagnosis of pulmonary tumors; however, cytologic specimens have only infrequently been used for molecular studies. In this study, we performed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay to detect theAPI2-MALT1 fusion transcript in archival cytologic specimens used as RNA sources. We studied 3 pulmonary MALT lymphoma cases that were positive for the fusion gene as detected with RNA extracted from diagnostic histologic specimens. In 1 case, a conventional PCR clonality assay for the immunoglobulin heavy chain gene rearrangement failed to detect the monoclonality. In all 3 cases, the fusion transcript was successfully detected in the cytologic specimens of sputum, bronchoalveolar lavage fluid, bone marrow smears, and pleural effusions. This finding suggests that such specimens can be used as RNA sources in multiplex RT-PCR assays for theAPI2-MALT1 fusion transcript. The detection ofAPI2-MALT1 fusion as carried out with these specimens would be useful as an ancillary assay for the diagnosis, staging, and follow-up of pulmonary MALT lymphoma.