Lymphocytes are called “nature's best known cells”, since their function has been studied in detail by many aggressive immunologists and their surface phenotypes have been investigated thoroughly with the monoclonal antibodies (MAbs) to surface antigens of lymphocytes. In addition, a surprisingly rapid progress of gene engineering, including that of immunoglobulin and T cell receptor has been used for the investigation of lymphocyte genotypes. This knowledge was directly applied for the analysis and diagnosis of lymphoid diseases.
Three in vitro human germ cell tumor cell lines with different cell lineage and variable capability of differentiation were established from testicular germ cell tumors. NCR-G1 corresponding to yolk sac tumor were induced α-fetoprotein (AFP) production with retinoic acid (RA) treatment. NCR-G2 and -G3 were human embryonal carcinoma (EC) cell lines. They have cytological and immuno-phenotypic characteristics common to human EC cells as has been described in other EC cells. NCR-G2 grew as floating cell aggregates in the culture medium, whereas NCR-G3 grew as floating cell aggregate and flattered cells attached to the surface of culture dish. These flattened cells of NCR-G3 immunohistochemically expressed a variety of differentiation antigens including myogenic markers, extraembryonic trophoblastic cell marker (human chorionic gonadotropin (hCG)), and extracellular proteins. Differentiation capabilities of NCR-G3 cells became more evident when they were treated with RA. By flowcytometric analysis, human EC cell surface markers 2D7, 2H2 and 5D4 which were mouse monoclonal antibodies we obtained from immunization by NCR-G2 cells, disappeared from cell surface of RA-treated NCR-G3 cells. Consistent with these findings, the flattened cells of NCR-G3 that attached to the culture dish were negative for these antibodies. Moreover, longer exposure to RA enhanced hCG production in exposure time dependent fashion. These observations clearly indicate that NCR-G3 possess multipotent differentiation capabilities and they differentiated into the trophoblastic cell lineage other than somatic cells. In contrast to NCR-G3, NCR-G2 did not show any differentiation capabilities with RA treatment. All three germ cell tumor lines produced tumors in athymic mice with 100% efficiency. High AFP content was observed in the sera of NCR-G1 tumor-bearing mice. High hCG and AFP contents were detected in the sera of the NCR-G3 tumor-bearing mice. Thus, our newly established human germ cell tumor cell lines add new insights of molecular mechanism of human embryogenesis and differentiation of EC cells.
Employing labeled lectins as a probe, we examined by light and electron microscopy histochemical aspects of the process of experimentally induced metastasis. Mouse colon carcinoma cells (colon 26) were injected into the spleen of Balb/c mice and liver metastasis was induced. Metastasized colon 26 cells firstly attached to the liver endothelial cells, especially to those of sinusoidal capillaries. More than 10 days after injection, colon 26 cells were found in the subendothelial tissue and/or Disse's space where they formed metastatic colonies. Such metastasized cells and small colonies of colon 26 cells revealed positive stainings with GS-1, VVA, HPA, and ECA, which indicate these lectins to be useful for the early screening of the formation of liver metastasis. Among them, ECA stained the metastasized colon 26 cells rather strongly compared with the heterogeneous and faint staining in non-metastasized tumor foci in the spleen or in the subcutaneous tissue. This finding suggests the possibility that the expression of ECA-binding sugar structures on the cell surface may be involved in the interaction between carcinoma cells and liver endothelium.
We have previously reported that abnormal accumulation of p53, an onco-suppressor gene product, is immunohistologically demonstrable in microwave (MW)-fixed, paraffin-embedded sections of colorectal tumors with the use of a monoclonal antibody, PAb1801 (Am. J. Clin. Pathol., 97: 244-249, 1992). This monoclonal antibody targets the human specific epitope of p53 molecule but cannot distinguish the mutant form from wild-type p53, and no immunoreactivity of a mutant-specific monoclonal antibody, PAb240, could be observed in MW-fixed sections. In the present paper, we have executed a comparative immunohistochemical study on p53 and 70Kd heat shock proteins (HSP70) in MW-fixed sections of 20 colorectal carcinomas and 20 adenomas because mutant p53 has been reported to form a stable complex with HSP70. The use of PAb1801 resulted in nuclear p53 being detected in 11 out of 20 carcinomas and p53 being expressed focally in five out of 20 adenomas. In the adjacent sections, the majority of p53-positive carcinomas and adenomas also expressed HSP70 in the nucleus as well as in the cytoplasm. Coincident expression of p53 and HSP70 in the nuclei of colorectal tumor cells implies that the p53 detected with PAb1801 may be the mutated protein that forms stable complexes with HSP70.
Fifty-one cases of human renal cell carcinoma were investigated for immunohistochemical localization of ras oncogenes and their products. The immunohistochemical results were evaluated comparatively with the histological grade, stage and progression of the tumor. The ras p21 oncogene product was positive mainly in the plasma membrane of the renal cell carcinomas. The tissues from normal kidneys were limitedly stained in the cytoplasm of the proximal convoluted tubule cells. Five of 28 (17.9%) frozen specimens of renal cell carcinoma revealed the positive staining for ras mRNA by means of in situ hybridization as well as the strongly positive staining for ras p21 gene products. These 5 cases were all grade 2 and pT2b in TNM classification. However, there was no point mutation on exon 1 (corresponding to codons 12 and 13) and exon 2 (corresponding to codon 61), where point mutations are reported in several human tumors. Positive staining cases for ras p21 (survival rate of 81.2% at 5 years) showed the tendency to decrease the survival rate compared to the ras p21 negative staining cases. There was a significant correlation of ras p21 positive staining and the pathological extent of the primary tumor (pT) (p<0.05). Our immunohistochemical study revealed that the expression of ras p21 is one of the parameters related to the prognosis of renal cell carcinomas.