Mechanisms involved in chemotherapy-induced apoptosis and their implications in cancer chemotherapy.

The mechanisms by which chemotherapeutic agents kill neoplastic cells have been controversial. Recently, however, accumulated evidence has suggested that these agents exert their cytotoxic effects mainly by inducing apoptosis in tumor cells. This article reviews the findings of recent studies on the mechanisms by which chemotherapeutic agents induce apoptosis and their implications in cancer chemotherapy.

TGF-beta 1 induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells.

TGF-beta1 inhibits the cell cycle progression of many types of cells by arresting them in the G1 phase. This cell cycle arrest has been attributed to the regulatory effects of TGF-beta1 on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of proteins, such as p15INK4b, p21WAF1/Cip1, and p27Kip1, that physically associate with cyclins, cyclin-dependent kinases (Cdk), or cyclin-Cdk complexes. In epithelial cell lines, TGF-beta1 was previously shown to inhibit cell cycle progression through down-regulation of Cdk4 and/or up-regulation of p15INK4b and/or p21WAF1/Cip1. However, TGF-beta1 had little or no effect on the p27Kip1 mRNA and protein levels. In this report, we show that, in contrast to observations in epithelial cell lines, TGF-beta1 increased the p27Kip1 mRNA and protein levels in the murine B cell lines CH31 and WEHI231. This TGF-beta1-mediated induction of p27Kip1 also resulted in an increased association of p27Kip1 with Cdk2 and a decreased Cdk2 kinase activity. In contrast to epithelial cells, however, TGF-beta1 had little or no effect on the Cdk4 and p21WAF1/Cip1 protein levels in these B cells. Finally, although several studies suggested a direct role of p53 in TGF-beta1-mediated cell cycle arrest in epithelial cells, TGF-beta1 inhibited cell cycle progression in CH31 even in the absence of wild-type p53. Taken together, these results suggest that TGF-beta1 induces G1 arrest in B cells primarily through a p53-independent up-regulation of p27Kip1 protein.

Expression of Bcl-x in T cells.

The thymus is a major site of apoptosis where programmed cell death is involved in the deletion of self-reactive T cells. We have investigated the role of bcl-x in T cells by defining the expression of its two isoforms, bcl-x and bcl-xs, in a series of human thymocyte cell lines and in human T lymphocytes using the ribonuclease protection assay. Bcl-x1 was the predominant isoform expressed in T cell lines and in T lymphocytes, where expression was further enhanced by PMA/ionomycin. This broad expression supports a central role for bcl-x in thymocyte development perhaps through post-transcriptional regulation.

TPA-induced arrest of erythroid differentiation is coupled with downregulation of GATA-1 and upregulation of GATA-2 in an erythroid cell line SAM-1.

GATA-1 protein is thought to be a positive regulator of erythroid differentiation. However, ectopic expression of a conditional GATA-2/estrogen receptor chimera was shown to inhibit erythroid differentiation in a hormone-dependent manner, suggesting the negative regulation of erythroid differentiation by GATA-2 protein. Accordingly, we reasoned that the quantitative balance of GATA-1 and GATA-2 protein might affect erythroid differentiation. In this report, we performed specific and quantitative measurements of GATA-1 and GATA-2 protein in a new erythroid cell line, SAM-1, after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). On the basis of these measurements, we show that TPA-induced arrest of erythroid differentiation is coupled with the upregulation of GATA-2 protein, as well as the downregulation of GATA-1 protein. Our results suggest that it is the precise quantitative balance of GATA-1 and GATA-2 protein that regulates erythroid differentiation.

Role of bcl-2 and IL-5 in the regulation of anti-IgM-induced growth arrest and apoptosis in immature B cell lines. A cooperative regulation model for B cell clonal deletion.

Recent studies of transgenic mice have confirmed that clonal deletion is involved in the development of B cells. However, little is known about intercellular and intracellular molecular events regulating B cell clonal deletion. We investigated the role of bcl-2 and cytokines in the regulation of B cell clonal deletion using anti-IgM-induced growth arrest and apoptosis in immature B cell lines as a model. We show here that overexpression of Bcl-2 protein in stably transfected immature B cells partially inhibits anti-Ig M-induced apoptosis but does not affect growth arrest. Similarly, IL-5 has a strong inhibitory effect on anti-IgM-mediated apoptosis but has a weak inhibitory effect on growth arrest. Finally, although both bcl-2 overexpression and exogenous IL-5 cooperate with bacterial LPS to block apoptosis, bcl-2 overexpression and exogenous IL-5 have no additive inhibitory effect on anti-Ig induced apoptosis. These findings indicate that anti-IgM-induced apoptosis is independently regulated from growth arrest and is controlled by at least two independent pathways: One is regulated by either Bcl-2 protein or IL-5 and the other is regulated by LPS. Activation of both the bcl-2/IL-5 and LPS pathways is necessary for complete inhibition of apoptosis, and presumably, clonal selection of the immature B cells.

bcl-2 protein inhibits etoposide-induced apoptosis through its effects on events subsequent to topoisomerase II-induced DNA strand breaks and their repair.

Previous studies have shown that bcl-2 overexpression can inhibit apoptosis induced by DNA-damaging agents widely used in cancer chemotherapy, including X-irradiation, alkylating agents (hydroperoxycyclophosphamide, etc.), and topoisomerase II inhibitors (etoposide, etc.). However, little is known about the mechanism by which bcl-2 overexpression inhibits apoptosis triggered by these agents. In this study, we examined whether bcl-2 overexpression could have effects on etoposide-induced DNA damage and its repair. For these experiments, we developed CH31 clones (mouse B-cells) stably transfected with human bcl-2 sense plasmids and compared these clones with a parental CH31 clone or CH31 clones with antisense plasmids. Overexpression of bcl-2 protein inhibited etoposide-induced apoptosis and cytotoxicity. However, there was no or little difference in the production and repair of DNA-protein cross-links, DNA single-strand breaks, and double-strand beaks among a parental CH31 clone and CH31 clones with human bcl-2 sense or antisense plasmids. These findings indicate that (a) apoptosis or cytotoxicity induced by etoposide can be separated into early events (formation of double-strand breaks, DNA single-strand breaks, and double-strand breaks) and later events (secondary DNA fragmentation or cell death) and (b) bcl-2 inhibits apoptosis and cytotoxicity induced by etoposide at some steps between these events.

Angiotropic lymphoma with histologic features of neoplastic angioendotheliomatosis presenting with predominant respiratory and hematologic manifestations. Report of a case and review of the literature [corrected].

Neoplastic angioendotheliomatosis (NAE) is a rare fatal disease characterized by widespread intravascular proliferations of neoplastic mononuclear cells. Clinically, dermatologic and bizarre neurologic manifestations usually predominate. The origin of the neoplastic cells remains still undetermined. The authors report a patient with NAE peculiar with respect to the following points: (1) the patient predominantly manifested respiratory symptoms and hematologic findings and lacked cutaneous or neurologic manifestations; and (2) immunohistochemical and molecular genetic studies showed the B-cell nature of the neoplastic cells, although previous cases with predominant respiratory or hematologic manifestations were reported to be of endothelial origin. Despite the rarity, this type of NAE or angiotropic [corrected] lymphoma should be recognized because it is easily confused with other disorders, particularly vasculitis or thrombotic thrombocytopenic purpura.

[Three cases of malignant lymphoma developing from chronic empyema wall].

Three patients with non-Hodgkin's lymphoma (NHL) involving the lung or chest wall are reported. All patients had tuberculous pleuritis or received artificial pneumothorax for pulmonary tuberculosis 30 years or more previously. The NHL of these patients developed in or close to the thickened pleura. Histologic examination showed diffuse large cell type (LSG classification) in all cases. Lymphomas of all cases were considered to have originated from B-cell lineage because their neoplastic cells expressed B1 (CD20) antigens. In no case the tumors were resectable, and chemotherapy or radiotherapy were performed. Two died of local NHL at 5 and 6 months respectively after diagnosis. One patient is still alive at 8 months after the first treatment for NHL. Early diagnosis is essential to improve the prognosis of these NHLs although it is difficult because of the co-existent pyothorax. Following features seemed to be useful for diagnosis: 1) chest pain or shoulder pain, 2) elevation of LDH level, 3) mass shadow in computed tomography, and 4) abnormal uptake of gallium 67. As precise assessment of tumor size is difficult because of the co-existent pyothorax and necrosis, follow-up studies by gallium scan or magnetic resonance imaging might be useful for proper treatment.

Molecular analysis of a chromosomal translocation, t(9;14)(p13;q32), in a diffuse large-cell lymphoma cell line expressing the Ki-1 antigen.

We have studied a translocation, t(9;14)(p13;q32), in a diffuse large-cell lymphoma cell line, KIS-1, that expresses the Ki-1 (CD30) antigen. Molecular cloning of the immunoglobulin heavy-chain locus (IGH) of this cell line revealed an unknown segment linked 5' to IGH. The breakpoint on chromosome 14 was 265 base pairs downstream from the 3' border of the JH6 joining gene segment. Class switch recombination deleted most of the constant genes of IGH (CH) and juxtaposed the C alpha 2 gene downstream of the translocation junction. Analysis of somatic cell hybrids and in situ chromosomal hybridization demonstrated that the translocated segment was normally located at band p13 of chromosome 9. The chromosome 9 sequences were transcriptionally active, giving rise to transcripts of approximately 11 kilobases. The KIS-1 cells seemed to have a small quantity of chimeric transcripts containing both chromosome 9 and C alpha 2 sequences.

Acute transformation of chronic large granular lymphocyte leukemia associated with additional chromosome abnormality.

A patient with large granular lymphocyte (LGL) leukemia that transformed into an acute or aggressive form after 20 months of the chronic phase is reported. The patient's leukemic cells were mature, medium-sized lymphocytes with sparse azurophil granules and the surface phenotypes of the cells were CD2+, CD3-, CD11+, and CD16+. Molecular analysis showed a germ line configuration in both T-cell receptor beta-chain genes and T-cell receptor tau-chain genes. A clonal anomaly of chromosome (trisomy 8) was demonstrated in peripheral blood cells. LGL after acute transformation of the disease displayed large blastic morphology with prominent nucleoli, intense basophilic cytoplasm, and numerous granules. Karyotypic analysis demonstrated a mosaic of trisomy 8 and trisomy 8 with an additional marker chromosome. Thus, transformation of chronic LGL leukemia into an acute or aggressive form in this patient was associated with morphologic and karyotypic changes of the leukemic cells. Patients with a stable form of chronic LGL leukemia should be examined carefully for the possible acute crisis associated with a clonal evolution.

[Successful management of massive hemoptysis, following pulmonary mycosis by bronchial artery embolization in a patient with acute lymphoblastic leukemia].

In June 1987, a 20-year-old man was diagnosed as T-cell acute lymphoblastic leukemia. In August, at a nadir period of the remission induction chemotherapy, he complained of high fever and dry cough. A chest roentgenogram also showed a nodular area of increased density in the left upper lobe. Since his clinical state deteriorated progressively despite the administration of broad-spectrum antibiotics, amphotericin B was administered intravenously (with an initial dose of 5 mg/day, increased up to 25 mg/day). Concomitant with bone marrow recovery and continued antifungal therapy, he became soon afebrile and improved over the next 2 months. The infiltrates also began to resolve. Then he abruptly coughed up about 800 ml of blood and suffered from acute respiratory failure. Bronchial arteriographic studies demonstrated active extravasation of contrast medium in the region of the cavity. After therapeutic embolization with Gelfoam, the extravasation was no longer observed. Active bleeding abruptly ceased and had not recurred until the left upper lobectomy which was performed 10 days after the embolization. This case typically demonstrates the value of bronchial arterial embolization in treating massive hemoptysis.

Immunohistochemical analysis of peripheral T-cell lymphoma in Japanese patients.

The authors immunohistochemically analyzed the phenotype of 40 cases of peripheral T-cell lymphoma, including 12 adult T-cell leukemia/lymphoma (ATL) cases. Molecular genetic analysis of the T-cell receptor beta-chain and immunoglobulin heavy chain genes were also applied to cases of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD)-like lymphoma and so-called Lennert's lymphoma. Twenty non-ATL lymphomas expressed a helper/inducer phenotype, whereas only one extranodal case expressed a suppressor/cytotoxic phenotype. Three cases had a CD4-CD8- phenotype, and two cases a CD4+CD8+phenotype. No specific relationship between morphologic characteristics (LSG classification) and phenotype was found among non-ATL lymphomas. Six of eight AILD-like lymphomas had a helper/inducer phenotype. Monoclonality of neoplastic T-cells was demonstrated in six of the seven cases of AILD-like lymphoma by molecular genetic analysis. Two cases of Lennert's lymphoma also showed a helper/inducer phenotype and rearrangement of the T-cell receptor beta-chain gene. Serologically defined ATL cases had a helper/inducer phenotype except in one case that expressed both CD4 and CD8. None of the ATL cases had the CD7 antigen in this study using WT 1 as a CD7 antibody, which is in contrast with the non-ATL lymphomas in which 13 of 25 cases expressed CD7. CD25, strongly detectable in all ATL cases, was negative or weakly expressed in non-ATL lymphomas. These facts suggest that non-ATL and ATL are in the different biologic state, probably resulting from the integration of human T-cell leukemia virus type I (HTLV-I), although both are derived from helper/inducer T-cells.

A novel B cell line established from Ki-1-positive diffuse large cell lymphoma.

A novel cell line, designated KIS-1, was established from a patient with Ki-1-positive diffuse large cell lymphoma. Multiple phenotypic analysis of the KIS-1 cells was carried out with a total of 22 monoclonal antibodies defining hematopoietic cell subsets and lineages. The KIS-1 cells were positive for Ki-1, B4, HLA-DR, and 2D1 (common leucocyte) antigens, but were negative for the antigens reportedly specific for T cells, natural killer cells, granulocytes, monocytes, interdigitating reticulum cells and dendritic reticulum cells. The genomic analysis of the KIS-1 cells showed not only the rearrangement of JH and J kappa genes but also the probable rearrangement of C lambda genes. Moreover, the cells produced immunoglobulin lambda chains. Thus, KIS-1 was considered to be of B-cell lineage. The lymphoma-cell derivation of KIS-1 was based on the following facts. The cytochemical, immunologic, cytogenetic properties and the results of the molecular genomic analysis in the KIS-1 cells were essentially the same as those of the original tumor cells, and the KIS-1 cells were negative for Epstein-Barr virus-associated nuclear antigen. KIS-1 is the only known B-cell line derived from Ki-1-positive diffuse large cell lymphoma, and should be useful for defining the biological implications of Ki-1 antigen.

Malignant histiocytosis with rearrangement of the heavy chain gene and evidence of monocyte-macrophage lineage.

A unique case of malignant histiocytosis (MH) is reported. Its origin from the monocyte-macrophage system was indicated by expression of highly specific myeloid cell markers (My4, MCS2, and cytoplasmic lysozyme), diffuse activity of acid phosphatase and NaF-sensitive alpha-naphthyl acetate esterase, lack of immunologic markers specific for other cell lineages, and germ line configuration of the immunoglobulin light chain gene and the T-cell receptor beta-chain gene. Its neoplastic nature was suggested by the single rearranged band of the immunoglobulin heavy chain gene.