Yoshiki Uemura

Circulating serpin tumor markers SCCA1 and SCCA2 are not actively secreted but reside in the cytosol of squamous carcinoma cells

An elevation in the circulating level of the squamous‐cell carcinoma antigen (SCCA) can be a poor prognostic indicator in certain types of squamous‐cell cancers. Total SCCA in the circulation comprises 2 nearly identical, ∼45 kDa proteins, SCCA1 and SCCA2. Both proteins are members of the high‐molecular weight serine proteinase inhibitor (serpin) family with SCCA1 paradoxically inhibiting lysosomal cysteine proteinases and SCCA2 inhibiting chymotrypsin‐like serine proteinases. Although SCCA1 and SCCA2 are detected in the cytoplasm of normal squamous epithelial cells, neither serpin is detected normally in the serum. Thus, their presence in the circulation at relatively high concentrations suggests that malignant epithelial cells are re‐directing serpin activity to the fluid phase via an active secretory process. Because serpins typically inhibit their targets by binding at 1:1 stoichiometry, a change in the distribution pattern of SCCA1 and SCCA2 (i.e., intracellular to extracellular) could indicate the need of tumor cells to neutralize harmful extracellular proteinases. The purpose of our study was to determine experimentally the fate of SCCA1 and SCCA2 in squamous carcinoma cells. Using subcellular fractionation, SCCA‐green fluorescent fusion protein expression and confocal microscopy, SCCA1 and SCCA2 were found exclusively in the cytosol and were not associated with nuclei, mitochondria, lysosomes, microtubules, actin or the Golgi. In contrast to previous reports, metabolic labeling and pulse‐chase experiments showed that neither non‐stimulated nor TNFα/PMA‐stimulated squamous carcinoma cells appreciably secreted these ov‐serpins into the medium. Collectively, these data suggest that the major site of SCCA1 and SCCA2 inhibitory activity remains within the cytosol and that their presence in the sera of patients with advanced squamous‐cell carcinomas may be due to their passive release into the circulation. Int. J. Cancer 89:368–377, 2000.

SCCA1 and SCCA2 Are Proteinase Inhibitors That Map to the Serpin Cluster at 18q21.3

The genes for the squamous cell carcinoma antigen (SCCA) were found flanking a deletion breakpoint from a patient with the 18q-syndrome. The genes are <10 kb apart, tandemly arrayed in a head-to-tail fashion, and∼10 kb in size. Both genes also contain 8 exons and identical intron-exon boundaries. The cDNAs encode for proteins that are 92% identical and 95% similar. Amino acid comparisons show that SCCA1 and SCCA2 are members of the high-molecular weight serine proteinase inhibitor (serpin) family. Physical mapping studies show that the genes reside within the 500-kb region of 18q21.3 that contains at least four other serpin genes. The gene order is cen-maspin (PI5), SCCA2, SCCA1, PAI2, bomapin (PI10), PI8-tel. Biochemical analysis of recombinant SCCA1 and SCCA2 proteins shows that SCCA1 is a potent cross-class inhibitor of papain-like cysteine proteinases such as cathepsins L, S and K, whereas SCCA2 is an inhibitor of chymotrypsin-like serine proteinases such as cathepsin G and mast cell chymase. These findings suggest that SCCA1 and SCCA2 are capable of regulating proteolytic events involved in both normal (e.g., tissue remodeling, protein processing) and pathologic processes (e.g., tumor progression).

Effect of serum deprivation on constitutive production of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells

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.

Acute lymphoblastic leukaemia relapsing with solid hepatic tumours after bone marrow transplantation

A 38-year-old man was diagnosed with Philadelphia chromosome-negative acute lymphoblastic leukaemia in February 1992. After achieving complete remission with chemotherapy, he underwent bone marrow transplantation from his human leucocyte antigen (HLA)-identical brother in October 1992. However, the disease recurred after 3 years. Complete remission was again induced by chemotherapy and he was given a second bone marrow transplant from the same donor in 1996. One and a half years later, the patient developed malaise and left upper abdominal tenderness. An enhanced computed tomography scan revealed two large mass lesions in the liver (top left). A percutaneous biopsy of the liver demonstrated the presence of primitive lymphoid cells. At that time, no leukaemic cells were seen in the peripheral blood or bone marrow. The hepatic lesions were thought to represent either recrudescence of leukaemia or post-transplantation lymphoma. To determine the diagnosis and to reduce the tumour burden, partial hepatectomy was performed in December 1997. The resected liver contained solid tumours with yellowish-white cut surfaces (bottom left). Histologically, the tumours were composed of lymphoblasts with frequent mitoses (right, haematoxylin and eosin); the immunophenotype (CD10, CD19, HLA-DR) was identical to that of the original leukaemic cells. Three weeks after hepatectomy, leukaemic cells appeared in the peripheral blood and bone marrow. Although the patient entered haematological remission with chemotherapy, meningeal and systemic relapses occurred, causing death in 1999.