Squamous cell lung carcinoma cells obtained from a patient who presented with leukocytosis and hypercalcemia were transplanted into nude mice anda serially transplantable cell line, OKa‐N‐1, was established. The nude mice transplanted with OKa‐N‐1 cells displayed leukocytosis and hypercalcemia. Serum levels of granulocyte colony‐stimulating factor (G‐CSF) and parathyroid hormone‐related protein (PTHrP) were both elevated in these mice. In vitro cultivation of this tumor cell line gave rise to a clonal cell line, OKa‐C‐1. Nude mice transplanted with the OKa‐C‐1 cell line also showed leukocytosis and hypercalcemia with high serum G‐CSF and PTHrP levels. The culture supernatant of OKa‐C‐1 contained high levels of G‐CSF and PTHrP. Immunohistochemical studies showed the expression of PTHrP in OKa‐C‐1 cells. Reverse transcription polymerase chain reaction revealed the presence of G‐CSF and PTHrP mRNA in this cell line. Dexamethasone treatment inhibited the transcription of G‐CSF and PTHrP genes. This new human squamous carcinoma cell line, OKa‐C‐1, would be useful for studying the mechanism of simultaneous production of G‐CSF and PTHrP and their control in cancer patients with leukocytosis and hypercalcemia.
An autopsy case of a 61 year old male with primary squamous cell carcinoma of the lung with associated marked leukocytosis and hypercalcemia is reported. High levels of serum parathyroid hormone-related peptide (PTHrP) and granulocyte colony stimulating factor (GCSF) were detected. The tumor cells distinctly showed positive cytoplasmic immunoreactions with anti-PTHrP and anti-GCSF antibodies. Marked granulocytosis and thin bony trabeculae lacking osteoblasts were observed in the vertebral bone. Calcium deposits were found in the proximal tubules of the kidneys. Infarcts were seen as a result of fibrin thrombosis of the splenic artery. The tumor was successfully transplanted into nude mice in which the high levels of serum PTHrP and GCSF were reproduced. These results indicate that the tumor simultaneously produced both PTHrP and GCSF causing the paraneoplastic syndromes of hypercalcemia and leukocytosis.
Summary.We report a 52-year-old man with chronic myelogenous leukaemia (CML) who developed hypercalcaemia in blast crisis. There was a high serum level of parathyroid hormone-related protein (PTHrP). The leukaemia cells secreted PTHrP into culture medium when cultured in vitro and were shown to express PTHrP mRNA by reverse transcription-polymerase chain reaction. Transplantation of the leukaemia cells into nude mice resulted in the production of Ph 1 chromosome-positive tumours which caused increased levels of calcium and PTHrP in the blood. These results indicated that the hypercalcaemic event in the patient was induced by PTHrP of CML cell origin.
Previously we have established a clonal squamous cell carcinoma cell line OKa‐C‐1 derived from lung cancer of a patient with marked leukocytosis and hypercalcemia. OKa‐C‐1 cells simultaneously produce granulocyte colony‐stimulating factor (G‐CSF) and parathyroid hormone‐related protein (PTHrP) at the single cell level and cause paraneoplastic syndromes in nude mice bearing the tumor. It is known that the production of G‐CSF and PTHrP is individually regulated by inflammatory cytokines in various malignant cells. To investigate the common factors in the regulation of G‐CSF and PTHrP production in OKa‐C‐1 cells, we examined the effects of some inflammatory agents [lipopolysaccharide (LPS), phorbol‐12‐myristate‐13‐acetate (PMA), tumor necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1) β and IL‐6] on G‐CSF and PTHrP production, by means of enzyme‐linked immunosorbent assay (ELISA), immunoradiometric assay (IRMA) and quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). TNF‐α or IL‐1β induced both G‐CSF and PTHrP production in the conditioned medium. TNF‐α synergized with IL‐1β to significantly increase G‐CSF production. In addition, TNF‐α and IL‐1β strongly induced G‐CSF mRNA with peaks at 2 and 6 h respectively. Although PTHrP production was also strongly induced by TNF‐α PTHrP mRNA expression was more strongly induced by PMA than by TNF‐α. Thus, TNF‐α and IL‐1β could be common factors that individually and synergistically regulate G‐CSF and PTHrP production in OKa‐C‐1 cells. Moreover, G‐CSF and PTHrP production could be not only transcriptionally, but also posttranscriptionally regulated by other factors.
Peripheral blood cells from a female patient with Ph1‐positive chronic myelogenous leukemia (CML) in blast crisis were serially transplanted in BALB/c nude mice for 16 passages. This in vivo cell line, designated CML‐N‐1, had Ph1chromosome abnormality and BCR gene rearrangement. The cells expressed CD11b, CD13, CD33, CD34, CD38, and HLA‐DR antigens until the 11th passage and subcutaneous tumors produced by these passages were composed of admixtures of immature and maturing cells that differentiated to basophils when cultured in vitro. From the 12th passage on, the tumors became composed mainly of immature cells expressing CD13, CD34, and HLA‐DR, and no longer differentiated to basophils even upon in vitro culture. In contrast to the vigorous proliferation in vivo, CML‐N‐1 cells from any passage failed to proliferate in vitro under standard liquid culture conditions with or without growth factors, such as granulocyte‐macrophage colony‐stimulating factor, granulocyte colony‐stimulating factor, monocyte colony‐stimulating factor, interleukin 3, interleukin 6 and stem cell factor. However, a continuously growing cell line, designated CML‐C‐1, was established by culturing CML‐N‐1 cells on feeder layers of mouse bone marrow stromal cells. This mouse bone marrow stromal cell‐dependent cell line showed immature cell morphology and expressed early myeloid phenotype positive for CD13, CD34, and HLA‐DR. These results indicate that mouse bone marrow stromal cells provide a certain growth factor(s) active on human leukemia cells.
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