We previously established two lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce abundant granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF). Inflammatory cytokines, tumor necrosis factor-alpha (TNF-α α α α) and interleukin ( ccasionally, leukocytosis is observed in patients with several cell types of carcinoma with an aggressive clinical course.

BACKGROUND. Gastrin-releasing peptide (GRP) is an autocrine growth factor in patients with small cell lung carcinoma (SCLC). The authors developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of SCLC cells in the peripheral blood and the pleural effusion using preproGRP mRNA as a target. METHODS. The current study was conducted to determine the utility of preproGRPspecific nested RT-PCR on the peripheral blood, bone marrow, and pleural effusion samples from 32 patients with SCLC, 39 patients with non-small cell lung carcinoma (NSCLC), 28 patients with nonmalignant pulmonary disease, and 20 healthy volunteers. The internal primers were designed to amplify a 244-base pair PCR product, a sequence encompassing exon 1 and exon 2 by the nested RT-PCR assay. RESULTS. Amplification of the preproGRP message was detected in SCLC cell lines (LU165, SBC1, SBC2, and SBC3) but not in other NSCLC cell lines (A549, ABC1, EBC1, and Oka-1). The SCLC cells (LU165) were detected in dilutions of tumor cells of up to 10 Ϫ7 in hematopoietic cells from healthy donors. The preproGRP mRNA was detected in 16 of 32 (50%) blood samples, 2 of 11 (18%) marrow samples, and in all 6 (100%) pleural effusion samples. Blood samples gave positive results in 11 of 19 (58%) patients with extensive disease compared with 5 of 13 (38%) patients with limited disease. In contrast, only 1 blood sample (2.6%) from a patient with lung adenocarcinoma gave a positive result among patients with NSCLC. No other samples of blood, bone marrow, and pleural effusion from patients with NSCLC and none of the blood samples from patients with nonmalignant diseases and healthy volunteers were positive. CONCLUSIONS. The current RT-PCR approach may be a sensitive and specific assay to detect SCLC cells in circulating blood as well as in pleural effusions from SCLC patients.

We previously established 2 lung cancer cell lines, OKa‐C‐1 and MI‐4, which constitutively produce an abundant dose of granulocyte‐colony stimulating factor (G‐CSF) and granulocyte macrophage‐colony stimulating factor (GM‐CSF). Many other cases with G‐CSF or GM‐CSF producing tumors have been reported up to the present. However, the biological properties of the overproduction of G‐CSF and GM‐CSF by tumor cells have not been well known. Several reports demonstrated the presence of an autocrine growth loop for G‐CSF and GM‐CSF in nonhematopoietic tumor cells. We showed that exogenous G‐CSF and GM‐CSF stimulated cell growth in a dose‐dependent manner in OKa‐C‐1 and MI‐4 cells. We could detect the presence of G‐CSF and GM‐CSF receptors in both cell lines by RT‐PCR analysis. We have previously shown that inflammatory cytokines, tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β enhance the expression of G‐CSF and GM‐CSF in the cell lines. However, the factors that regulate constitutive production of G‐CSF or GM‐CSF by tumor cells are still unknown well. In our study, we first reported that serum deprivation stimulated constitutive production of G‐CSF and GM‐CSF by lung tumor cells through activation of nuclear factor (NF)‐κB and p44/42 mitogen‐activated protein kinase (MAPK) pathway signaling. We suggest that G‐CSF and GM‐CSF constitutively produced by tumor cells could grow tumor itself and rescue tumor cells from the cytotoxicity of serum deprivation. © 2004 Wiley‐Liss, Inc.

Some cancer patients manifest leukocytosis without overt inflammation. The production of granulocyte colony-stimulating factor (G-CSF) by the tumor itself is thought to be responsible for this paraneoplastc syndrome.1-3) At least 80 cases of G-CSF-producing tumors have been reported up to the present. Although many types of G-CSF-producing tumors such as carcinoma of the thyroid, 4, 5) kidney, 6) oral cavity, 7) bladder, 8) gallbladder, 9) stomach, 10) hepatoma 11) and melanoma 12) have been reported, carcinoma of the lung [13][14][15][16][17] is predominant. Large cell carcinoma is the overwhelmingly dominant histologic type in lung cancer. 18,19) However, there have been few reports confirming the establishment of a G-CSF-producing cell line; to our knowledge, 10 G-CSF-producing lung cancer cell lines have been reported. Four of them are derived from large cell carcinoma. 14,[20][21][22] Recently, we studied a patient with large cell lung carcinoma who exhibited leukocytosis, and we established a new cell line in vitro from this patient. This cell line produces a high level of G-CSF in the medium. In this paper, we describe the establishment and characterization of this new lung cancer cell line. MATERIALS AND METHODS PatientThe patient was a 69-year-old male with large cell carcinoma of the lung. He underwent left upper lobectomy. Histologically, the surgical specimen of the lung tumor showed large cell undifferentiated carcinoma. However, the tumor metastasized to Virchow's node after 4 years. Chemotherapy and radiotherapy afforded only temporary control and the tumor grew rapidly. As the disease progressed, his peripheral blood white blood cell (WBC) count increased to 92 000/mm 3 with 95% mature neutrophils, without evidence of infection. The serum level of G-CSF was 141 pg/ml (normal <30 pg/ml). The patient gradually deteriorated with cachexia, and died 9 months after recurrence. At autopsy, metastatic tumors were found in the bilateral lungs, anterior mediastinum, right adrenal gland and retroperitoneal lymphnodes, besides Virchow's node. Furthermore, he had a large quantity of malignant pleural effusion and ascites. Cell culture Tumor cells were obtained from the pleural effusion and cultured for the establishment of a cell line.