Apoptosis is a morphologically distinct set of cell death process, discriminated from necrosis, and is induced through the activation of intrinsic cell-suicide program. To understand better the fundamental process of apoptosis, which is expected to promote us to manipulate cell death to our advantages, cellular analysis of cell death is required since apoptotic cells often co-exist with necrotic ones in tissues. In this study, we attempted several histochemical trials to define a difference between apoptosis and necrosis using hydrocortisone-treated rat thymus and CCl₄-treated rat liver as models of apoptosis and necrosis, respectively. When the fresh frozen sections were stained for acid phosphatase activity, the staining in necrotic hepatocytes was lost, while that in apoptotic thymocytes was well retained, reflecting the integrity of lysosome in apoptosis. In situ nick translation (ISNT) as well as TUNEL, which detect DNA strand breaks, also allowed us to differentiate apoptotic cells from necrotic cells in the frozen sections; the apoptotic nuclei were stained only when the sections were treated with proteinase K (PK), whereas the necrotic nuclei were stained without PK treatment. However, in paraffin sections apoptotic nuclei were stained by these methods without PK treatment. Therefore, the criterion to discriminate both types of cell death did not hold in this case. We also addressed whether the expression of Fas or Fas ligand is a useful histochemical marker of apoptosis. Consequently, a positive correlation was confirmed in ischemia-reperfusion mouse kidney and in ulcerative colites, while it could not be always generalized. Thus, in fact any one of these methods is not absolute singly, but rather mutually supplementary. Currently, for a precise description of cell death, it seems essential to combine various methods including morphological and biochemical approaches.

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Cell proliferation and programmed cell death play an important roles in the maintenance of tissue dynamics in both physiologic and pathologic status. Increased proliferation and/or decreased programmed cell death or apoptosis can result in “abnormal proliferation”. On the other hand, decreased proliferation and/or increased apoptosis may cause “loss of the cells”. Therefore, simultaneous analysis of both cell proliferation and apoptosis can provide important information on understanding physiology and pathology of various tissues. Recent advent in immunohistochemistry including ki67 immunostain with microwave irradiation for antigen retrieval and MIB-1 monoclonal antibody and development of 3'-OH nick end labeling technique or TUNEL method made it possible to assess cell proliferation and apoptosis in formalin-fixed and paraffin-embedded materials with reasonable accuracy. We applied these methods to human adrenal cortex and its disor-ders and gastric mucosa with or without atrophy or intestinal metaplasia in order to understand tissue dynamics. In the human adrenal glands, apoptosis appears to play important roles in the maintenance of organized cortical cell turnover, i.e., centripetal movement of the cortical cells or cell proliferation in the outer fasciculata and cell death in the zona glomerulosa and the zona reticularis, and in tumor cell dynamics in adrenocortical neoplasms. In non-neoplastic human gastric mucosa, marked increase of the number of the cells positive for both Ki67 and 3'-OH nick end labeling was observed in the gastric glands associated with incomplete intestinal metaplasia. In addition, marked mRNA hybridization signals of p53 was detected in the cells undergoing apoptosis in these glands. Gastric glands associated with incomplete intestinal metaplasia are therefore characterized by increased cells turnover and possibly p53 induced apoptosis.

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Using human erythroleukemia cell line, TF₁, and bcl 2 cDNA transfected TF₁-bcl 2 cells, (1) the sequential change of apoptosis, (2) the relationship between differentiation and apoptosis and (3) cell cycle change were analyzed. When GM-CSF was depleted, TF₁ showed apoptosis as detected with May-Giemsa staining, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method as well as DNA ladder formation. This apoptosis was delayed only with spermine, but not zinc ion nor cycloheximide. The usage of specific inhibitors of tyrosine kinase and protein kinase C, herbimycin A and calphostin C, respectively, reveals that both tyrosine kinase and protein kinase C pathways are important for the survival and proliferation of TF₁ cells.
In order to explain the close relationship between differentiation process and apoptosis, bcl 2 cDNA, which is known to confer cells apoptosis-resistance, was transfected into TF₁ cells. TF₁-bcl 2 cells shows resistance to apoptosis induced with GM-CSF depletion and have the similar differentiation potentials as those of original TF₁ cells when induced with erythropoietin or TPA. These results suggest that apoptosis is not necessarily accompanied with differentiation process.
When GM-CSF was depleted, both cells showed G₁/S block accompanied with the gradual appearance of the apoptic population only in TF₁ cells. With the re-addition of GM-CSF after 24 hours depletion of GM-CSF, 8 hours delay to enter cell cycle in TF₁-bcl 2 cells was observed, suggesting that TF₁-bcl 2 stayed in G₀ phase.
The relationship between apoptosis and cell cycle of these two cell lines prompted us to analyze what the real cause was in apoptic process. Using GM-CSF depleted TF₁ and TF₁-bcl 2 cells, sequential changes of cyclins, cyclin inhibitors, early immediate genes such as myb and myc, death related genes, ICE and ICH, were analyzed. Unfortunately, our present experiments sheowed there wan no apparent significant difference between these two cell lines during GM-CSF depletion.

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The concept of apoptosis was originally proposed by Kerr et al. in 1972. Its morphological features involve nuclear and cytoplasmic condensation, apoptotic body and rapid uptake by phagocytes. This form of cell death effects single cells with living tissue without disruption of either architecture or function. The process of apoptosis avoids both the release of intracellular contents into the tissue and the resulting inflammation which characterizes necrosis. Apoptotic phenomenon was recently confirmed by biochemical and immunohistological techniques such as electrophoresis and the TUNEL method for DNA fragmentation.
We used 36 cases of lymphadenitis including necrotizing lymphadenitis (24 cases), abscess forming lymphadenitis (3 cases), tuberculous lymphadesitis (3 cases), tularemia (3 cases), non-specific lymphadenitis (3 cases) and 140 cases of non-Hodgkin's lymphomas (NHLs).
In necrotizing lymphadenitis, apoptotic cells which correspond to CD 4⁺ lymphocytes are localized at the necrobiotic focus (7.9%). Fas antigen is observed in blastoid cells (CD 8⁺) and Fas-ligand is present in macrophages. In addition, we detected human herpesvirus 6 (HHV 6) particles, antigens and genomes within phagocytized cells. These findings suggest that apoptosis in necrotizing lymphadenitis may be induced by virus or non-bacterial agents rather than the Fas/Fas-ligand system.
In the high-grade group of NHLs, c-myc and p-53 oncogens are highly expressed but bcl-2 protein is lower. C-myc positive NHLs are significantly rich in TUNEL-positive (apoptotic) cells. In addition, it is known that bcl-2 protein tends to impede apoptosis mediated by c-myc oncogen.
Our data show that the coexpression of c-myc and p-53 oncogens may induce apoptosis rather than Fas and bcl-2 oncogens because of a lower inhibitory effect of bcl-2 protein in the high-grade group.
Apoptosis is an important cell regulator in tissues under both physiological and pathological conditions. The induction mechanism of apoptosis is different among reactive diseases and malignancy and it requires intimate studies from the standpoint of molecular biological aspects.

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Profound immunodeficiency in multicentric Castleman's disease (MCD) has already been indicated.
We evaluated immune function in four MCD patients. These results show: (1) polyclonal B cell activation in 4/4 patients, (2) decreased T cell colony formation in 3/4 patients, (3) decreased NK activity and NK cell number in 2/4 patients, (4) spontaneous IL-6 production in peripheral blood monocyte. These results showed that MCD resemble acquired immunodeficiency syndrome (AIDS).
Moreover, we indicated increased T cell activation in MCD, with this activation probably resulting in apoptosis. T cell apoptosis may be related to the pathogenesis of MCD.

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It is known that adult T-cell leukemia (ATL) cells undergo spontaneous apoptosis in vitro as a results of expression of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. However, the molecular mechanism of the apoptosis remained to be elucidated. We examined the expression of Fas-Ag, Bcl-2 and Tax in ATL cells prior to culture and following culture. ATL cells from some patients showed a increased level of Fas-Ag and a decreased Ievel of Bcl-2 compared to peripheral Iymphocytes from healthy individuals. Culture of ATL cells resulted in apoptosis. However, the apoptosis was confined to cells associated with a high level of Tax and a decreased level of Bcl-2. In addition, we found that apoptosis induced by HTLV-I Tax results in destruction of the HTLV-I genome itself. These results suggested that decreased Bcl-2 may be important in Tax-induced apoptosis.

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Resident macrophages in hematopoietic tissues, which are derived from precursor cells in the bone marrow, are components of the stroma. These macrophages are clearly differentiated from reticular cells or endothelial cells by the macrophage-specific antigen. Erythroblasts adhere to these macrophages via VLA-4-VCAM-1 and proliferate and differentiate in the presence of erythropoietin. Each macrophage that adhered to erythroblasts extends cytoplasmic processes so as to enfold erythroblasts, resulting in the formation of an “erythroblastic island”. This type of interaction between hematopoietic cells and macrophages via adhesion molecules may be important in the regulation of hematopoiesis.

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The clinical features and therapeutic effects were investigated in 184 patients with extranodal lymphoma among 491 patients with non-Hodgkin's lymphoma (37.5%). Of the 184 patients, 85 had their primary lesions in the gastrointestinal tract, 37 in Waldeyer's ring, 24 in the paranasal sinus, 10 in the central nervous system, 5 each in the testis and skin, 4 in the thyroid, 3 each in the lung and salivary gland, 2 each in the adrenal, spleen, and muscle, and 1 each in the bone and mammary gland. Histopathology showed that the lymphoma was of low grade in 8 patients, intermediate grade in 169, and high grade in 7. There were 134 B cell lymphoma and 32 T cell lymphoma. A complete remission (CR) was achieved in 90.8% of the patients. The overall 5-year survival rate was 62.5% and the disease-free 5-year survival rate was 60.9%. Prolonged survival was achieved in patients having the primary lesion in Waldeyer's ring, the stomach, or the skin, while the outcome was poor for patients with the primary in the central nervous system or testis. Multivariate analysis showed that the factors associated with prognosis were LDH, stage, histological type, CRP, and bone marrow invasion. The site of the primary lesion was also a prognostic factor. More effective therapy is needed for the patients with their primary lesions in the central nervous system and testis.

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