Munehisa Imaizumi

Genetic alteration of the β-catenin gene (CTNNB1) in human lung cancer and malignant mesothelioma and identification of a new 3p21.3 homozygous deletion

The β-catenin gene (CTNNB1) has been shown to be genetically mutated in various human malignancies. To determine whether the β-catenin gene is responsible for oncogenesis in thoracic malignancies, we searched for the mutation in 166 lung cancers (90 primary tumors and 76 cell lines), one blastoma and 10 malignant mesotheliomas (two primary tumors and eight cell lines). Among the lung cancers, including 43 small cell lung cancers (SCLCs) and 123 non-small cell lung cancers (NSCLCs), we identified four alterations in exon 3, which is the target region of mutation for stabilizing β-catenin. One primary adenocarcinoma had a somatic mutation from C to G, leading to an amino acid substitution from Ser to Cys at codon 37. Among the cell lines, SCLC NCI-H1092 had a mutation from A to G, leading to an Asp to Gly substitution at codon 6, NSCLC HCC15 had a mutation from C to T, leading to a Ser to Phe substitution at codon 45, and NSCLC NCI-H358 had a mutation from A to G, leading to a Thr to Ala substitution at codon 75. One blastoma also had a somatic mutation from C to G, leading to a Ser to Cys substitution at codon 37. Among the 10 malignant mesotheliomas, we identified a homozygous deletion in the NCI-H28 cell line. Cloning of the rearranged fragment from NCI-H28 indicated that all the exons except exon 1 of the β-catenin gene are deleted and that the deletion junction is 13 kb downstream from exon 1. Furthermore, Northern blot analysis of 26 lung cancer and eight mesothelioma cell line RNAs detected ubiquitous expression of the β-catenin messages except NCI-H28, although Western blot analysis showed that relatively less amounts of protein products were expressed in some of lung cancer cell lines. Our findings suggest that the β-catenin gene is infrequently mutated in lung cancer and that the NCI-H28 homozygous deletion of the β-catenin gene might indicate the possibility of a new tumor suppressor gene residing in this region at 3p21.3, where various types of human cancers show frequent allelic loss.

Characteristics of three human gastric cancer cell lines, NU-GC-2, NU-GC-3 and NU-GC-4.

Three human gastric cancer cell lines, NU-GC-2, NU-GC-3 and NU-GC-4 were establishedin vitro from the cancer tissues obtained from 3 patients during surgery. The pathological findings of the gastric tumors of these cases revealed poorly differentiated adenocarcinoma (and partial signet-ring cell carcinoma in the case of NU-GC-4). NU-GC-2 and NU-GC-4 were originally obtained from metastatic paragastric lymph nodes and NU-GC-3 was obtained from a metastatic tumor in the brachial muscle. The cells of NU-GC-2 and NU-GC-3 are polygonal in shape and grow as a monolayer sheet. NU-GC-4 cells, however, are mainly spherical in shape with a few free floating cells. Electron microscopy revealed epithelial characteristics in all 3 cell lines. The average doubling time of NU-GC-2 was 36.1 hours, that of NU-GC-3 was 38.2 hours and that of NU-GC-4 was 29.9 hours. The modal chromosome number of NU-GC-2 was 62, that of NU-GC-3 was 58 and those of NU-GC-4 grown inin vitro andin vivo were 52–54 and 53, respectively.In vitro andin vivo lines of NU-GC-4 were established from the same tumor. These two cell lines are quite similar in morphology, but slightly different in karyotype. Thein vitro sensitivity to anticancer agents was highest in NU-GC-4 and lowest in NU-GC-2. Of the anticancer agents, mitomycin C and adriamycin were most effective on the cells of all 3 cell lines.

Surgical indications for resection in pulmonary metastasis of choriocarcinoma

One hundred and twenty‐two patients with choriocarcinoma were treated from 1965–1977. Pulmonary metastasis was noted in 82 of the 122 patients. In 21 cases, open thoracotomy and lobectomy were performed in conjunction with chemotherapy. Fifteen patients achieved complete remission and six patients died. It became evident that the factors with the greatest effect on the outcome of the surgical treatment are the preoperative hCG values and the extent of pulmonary metastasis. The following are the authors established criteria for the operative intervention of pulmonary metastasis of choriocarcinoma (modifying Thomfords principles): (1) The patient must be a good risk for surgical intervention. (2) That the primary malignancy is controlled, (the uterus has already been resected, or no angiographical evidence of tumor in pelvic cavity). (3) There is no evidence of metastatic disease elsewhere in the body. (4) Roentgenologic evidence of pulmonary metastasis is limited to one lung. (5) The urinary hCG value is below 1000 miU/ml.

Evaluation of γ‐enolase as a tumor marker for lung cancer

The α‐enolase and γ‐enolase in tumor tissues and sera of patients with lung cancer were determined with an enzyme immunoassay system. Tissue γ‐enolase in small cell carcinoma of the lung (SCCL, n = 11), large cell carcinoma (n = 11), and non‐SCCL (except for large cell carcinoma) (n = 34) were enhanced approximately 35‐fold, ninefold, and fourfold, respectively; tissue γ/α + γ value of SCCL was significantly higher than that of normal lung tissue (P < 0.01). Serum γ‐enolase level was elevated (>6.0 ng/ml) in 14/18, 3/10, and 15/60 patients with SCCL, large cell carcinoma, and non‐SCCL (except for large cell carcinoma), respectively, and serum γ/α + γ value of SCCL was significantly higher than that of healthy subjects (P < 0.01). Immunohistochemically, the γ‐enolase was positive in 29/31 of the lung cancers. Serum γ‐enolase value is a useful tumor marker for staging and monitoring treatment of patients with lung cancer, and serum γ/α + γ value may be useful for differential diagnosis of SCCL from non‐SCCL or in differentiating lung cancers possessing neuroendocrine features from other lung cancers.

A case of multiple leiomyomatous lesions of the lung : an analysis of flowcytometry and hormone receptors

A 36 year old woman was admitted to our department because of a chest X-ray which showed multiple developing shadows. She underwent bilateral exploratory thoracotomies and a total 5 tumors were resected and pathologically diagnosed as benign metastasizing leiomyoma, the largest of which was positive for the progesterone receptor and negative for the estrogen receptor. A histogram of this tumor using a flow cytometer showed a diploid pattern and 4.6 percent of the S phase which was not more than that of a leiomyoma of the uterus from another patient. Two months later, she underwent a hysterectomy and bilateral salpingo-oophorectomy for treatment of the positive progesterone receptor in the pulmonary lesions. The resected uterine myoma and normal myometrium showed positive estrogen and progesterone receptors. For the subsequent 28 months she has been free of any further symptoms. Benign metastasizing leiomyoma of the uterus is a rare disease and very interesting because of its histological benignity and hormonal dependency. However, according to the literature, it is often confused in entity due to the fact that normal lung tissue also possesses hormone receptors. Considering our data on hormone receptors, it is rational to think that multiple leiomyomatous lesions in the lung should only be diagnosed as benign metastasizing leiomyomas when they possess positive estrogen and progesterone receptors.

Role of tyrosine specific phosphorylation of cellular proteins, especially EGF receptor and p125FAK in human lung cancer cells

To clarify the role of tyrosine phosphorylation of cellular proteins in human lung cancer cells, phosphotyrosine (PTYR)-containing proteins in lung cancer cell lines and in paired tissues resected from cancerous and normal lungs were studied by immunoblotting with an anti-PTYR antibody. We found that the profiles of protein phosphorylation were very similar among those cell lines which had different histological features. The major PTYR-containing proteins (180-190 KDa, 120-130 KD, and 95-100 KDa) were detected in lung cancer cell lines. The expression of EGF receptor (EGF-r) (p185) and o-erb B2 protein, and tyrosine phosphorylation of p125FAK were examined in cancerous lung tissues and normal lung tissues. In surgical specimens, approximately half of the samples of lung cancer tissues showed clear elevation of tyrosine phosphorylation. In these cancerous tissues, no clear amplification of EGF-r and c-erb B2 protein expression was observed. However, elevation of tyrosine phosphorylation of p125FAK was observed in cancerous lung tissues but not in normal lung tissues, and its phosphorylation was closely correlated with the nodal involvement of cancer and disease-free survival time. These results suggested that the intracellular signaling pathway via tyrosine phosphorylation plays a role in the generation and immortalization of lung cancer, and assessment of tyrosine phosphorylation of cellular proteins. especially p125FAK, may be available clinically as a prognostic factor.

Loss of heterozygosity of chromosome 12p does not correlate with KRAS mutation in non‐small cell lung cancer

Activating mutations of RAS gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. However, several studies have shown a frequent loss of the wild‐type KRAS allele in the tumors of murine models and an inhibition of oncogenic phenotype in tumor cell lines by transfection of wild‐type RAS, indicating that wild‐type RAS may have oncosuppressive properties. To determine whether loss of wild‐type KRAS is involved in the development of human lung cancer, we investigated the mutations of KRAS, NRAS and BRAF in 154 primary non‐small cell lung cancers (NSCLCs) as well as 10 NSCLC cell lines that have been shown to have KRAS mutations. We also determined the loss of heterozygosity status of KRAS alleles in these tumors. We detected point mutations of KRAS in 11 (7%) of 154 NSCLCs, with 10 cases at codon 12 and 1 at codon 61, but no mutations of NRAS or BRAF were found. Using the laser capture microdissection technique, we confirmed that 9 of the 11 tumors and 7 of the 10 NSCLC cell lines retained the wild‐type KRAS allele. Among the cell lines with heterozygosity of mutant and wild‐type KRAS, all of the cell lines tested for expression were shown to express more mutated KRAS than wild‐type mRNA, with higher amounts of KRAS protein also being expressed compared to the cell lines with a loss of wild‐type KRAS allele. In addition, among 148 specimens available for immunohistochemical analysis, 113 (76%) showed positive staining of KRAS, indicating that the vast majority of NSCLCs continue to express wild‐type KRAS. Our findings indicate that the wild‐type KRAS allele is occasionally lost in human lung cancer, and that the oncogenic activation of mutant KRAS is more frequently associated with an overexpression of the mutant allele than with a loss of the wild‐type allele in human NSCLC development.

A cell line from a human salivary gland mixed tumor

A cell line called Nagoya‐78 has been established from a human salivary gland mixed tumor. The cytoplasm of the cells contain much glycogen but little acid polysaccharide and mucin. The ultrastructure of the cells exhibit epithelial characteristics with desmosomes, and no virus particles are present. The doubling time is around 24 hours. The cell line can be cultured as a suspension, but the growth rate is lower than that of stationary cultures. The chromosome constitution approximates trisomy with 5 marker chromosomes. The cells grow slowly in the cheek pouch of unconditioned hamsters.

Immunochemical and immunohistochemical studies on three aldolase isozymes in human lung cancer

The aldolase isozymes A, B, and C in tumor tissues (63) and sera (104) of patients with lung cancer were determined with an enzyme immunoassay system, compared with normal lung tissues (13), and the sera of normal healthy subjects (100). Tissue aldolase A and C concentrations were enhanced in 83% (52/63) and 51% (32/63) of patients with lung cancer, respectively, regardless of histologic type or stage (P < 0.01). But aldolase B was not elevated in tissue levels. In the sera of patients with lung cancer, there were no significant elevations of the isozymes. Immunohistochemically aldolase A and C stained more intensely in the cytoplasm of lung cancer cells than those in normal tissues. These results indicate lung cancer cells contain enhanced tissue levels of aldolase A and C.

β-catenin inhibits cell growth of a malignant mesothelioma cell line, NCI-H28, with a 3p21.3 homozygous deletion

We have found that a malignant mesothelioma cell line, NCI-H28, had a chromosome 3p21.3 homozygous deletion containing the β-catenin gene (CTNNB1), which suggested that the deletion of β-catenin might have a growth advantage in the development of this tumor. To determine whether β-catenin has a growth-inhibitory activity, we transfected wild-type β-catenin, Ser37Cys mutant β-catenin as an activated type, and C-terminus deletion mutant β-catenin that lacks the transcription activity, into the NCI-H28 cells. A non-small cell lung cancer cell line, NCI-H1299, which expressed endogenous β-catenin, was also studied. We tested the localization of exogenous β-catenin in the NCI-H28 cells with immunofluorescence, and found that the wild-type β-catenin and the C-terminus deletion mutant were more strongly expressed in the plasma membrane and cytoplasm than in the nucleus, while the Ser37Cys mutant was more in the nucleus than in the cytoplasm. By using luciferase-reporter assay, the β-catenin/T-cell factor 4-mediated transactivity of the Ser37Cys mutant was shown to be higher than that of the wild-type β-catenin in both cell lines. However, the transactivity of the C-terminus deletion mutant was strongly reduced in both. Colony formation of the NCI-H28 cells was reduced by 50% after transfection with the wild-type β-catenin, and 60% with the Ser37Cys mutant, but only 20% with the C-terminus deletion mutant compared to the vector control. Inhibition of colony formation in NCI-H28 cells was because of apoptosis, manifested by positive staining of Annexin V and TUNEL assays in transfected cells. In contrast, when transfected with the wild-type β-catenin, no significant reduction in colony formation was seen in β-catenin wild-type NCI-H1299 cells. In conclusion, our data indicate that inactivation of β-catenin by a 3p21.3 homozygous deletion might be a crucial event in the development of the mesothelioma NCI-H28 cells. Thus, while β-catenin is well known to be a positive growth-stimulating factor for many human cancers, it can also act as a potential growth suppressor in some types of human cancer cells.