Monitoring of Heat Shock Response and Phenotypic Changes in Hepatocellular Carcinoma After Heat Treatment.

BACKGROUND/AIM: Local recurrence of hepatocellular carcinoma (HCC) after thermal coagulation therapy may be associated with an aggressive phenotypic change. This study focused on the thermal effects on HCC cells and evaluated the heat shock response and phenotypic changes after heat treatment. MATERIALS AND METHODS: HepG2 and HuH7 cells were used. After heat treatment at 37-50°C for 5-30 min, we assessed their survival rate, induction of heat shock protein (HSP)70 promoter, proliferation rate, induction of the epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-related markers. RESULTS: Induction of HSP70 promoter per surviving cell was maximized after 10 min of heat treatment at 48°C. Induction of EMT and CSC-related markers was also observed. CONCLUSION: Sub-lethal heat treatment causes large heat shock response to surviving HCC cells and induce EMT-like and CSC-like phenotypic changes that might contribute to increased aggressiveness.

Comparison of neuropathogenicity of poliovirus type 3 in transgenic mice bearing the poliovirus receptor gene and cynomolgus monkeys.

To clarify the similarities of poliovirus infection in cynomolgus monkeys and transgenic mice bearing the poliovirus receptor, TgPVR21, we compared the pathological changes of these animals following intraspinal inoculation of two strains of poliovirus type 3 using immunohistochemical detection of the capsid antigen. All of the monkeys inoculated with 10(6) TCID(50) viruses showed flaccid paralysis 2 or 3 days post-inoculation (p.i.). TgPVR21 mice showed paralysis starting from 2 to 3 days p.i. Histologically, neurons having pyknotic nuclei and eosinophilic cytoplasm and neuronophagia were characteristically observed in both animals, but central chromatolysis was not observed in infected TgPVR21. The median lesion scores in the monkeys and TgPVR21 were well correlated, though the distribution of poliovirus-infected lesions in the central nervous system was different. In both animals the motor neurons and the brainstem nuclei responsible for flaccid paralysis were infected by the virus, while the cerebral cortex and thalamus were infected in the monkeys but not in TgPVR21. These results confirmed the reliability of neurovirulence tests using TgPVR21 as a substitute for monkeys, in respect to the spinal and brainstem lesions of poliovirus type 3.

Human bone marrow stroma-dependent myeloma sister cell lines MOLP-6 and MOLP-7 derived from a patient with multiple myeloma.

Human bone marrow stroma (BST)-dependent myeloma sister cell lines MOLP-6 and MOLP-7 were established from the peripheral blood of a multiple myeloma (MM) patient with IgA kappa type MM (stage IIIB). The growth of the cell lines is constitutively dependent on BST cells; none of the cytokines tested nor the culture supernatant of the BST cells could support the growth. Both cell lines showed typical plasma cell morphology with abundant cytoplasm and one to four nuclei under Wright staining. The immunoprofiles of MOLP-6 and MOLP-7 correspond to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) chains, a heavy and kappa light chains, CD9, CD28, CD40, CD44, CD45, CD56, and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte associated markers. Both cell lines also expressed adhesion molecules including HCAM (CD44), VLA-4 (CD49d/CD29), VLA-6 (CD49f/CD29), ICAM-1 (CD54), NCAM (CD56), LFA-3 (CD58) and L-selectin (CD62L). The doubling time of MOLP-6 and MOLP-7 was 48 and 168 hours, respectively. In addition to this growth characteristic, the maximum cell density of each cell line was obtained at 1.7 x 10(6) cells/ml and 9.7 x 10(5) cells/ml, respectively. The characteristics of each cell line may reflect intraclonal variation of the proliferative capacity. The MOLP-6 together with the MOLP-7 sister will be useful model systems for the investigation of the biology of myeloma.

Human bone marrow stroma-dependent cell line MOLP-5 derived from a patient in leukaemic phase of multiple myeloma.

The novel multiple myeloma (MM) cell line MOLP-5 and its homologous sister cell line B407, a lymphoblastoid cell line (LCL), were established from the peripheral blood of a 71-year-old Japanese patient with Bence-Jones kappa-type multiple myeloma (stage IIIB with hyperammonaemia and hypercalcaemia). The growth of MOLP-5 cells is constitutively dependent on bone marrow stroma (BST) cells; none of the cytokines tested nor the culture supernatant of the bone marrow stroma cells could support the growth of MOLP-5. Wright-Giemsa-stained MOLP-5 cells showed typical plasma cell morphology with abundant cytoplasm and one to three nuclei. The immunoprofile of MOLP-5 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) kappa light chain, CD28, CD29, CD38, CD40, CD44, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA-1; the cells were negative for surface Ig and various other B-cell, T-cell and myelomonocyte-associated immunomarkers. Interleukin 6 (IL-6) receptor mRNA was found in the reverse transcriptase polymerase chain reaction (RT-PCR) analysis. IL-6 and IL-10 could induce cellular proliferation in short-term induction experiments. IL-6 or IL-10 production was not detected by specific enzyme-linked immunoabsorbent assay (ELISA). MOLP-5 cells expressed parathyroid hormone-related protein (PTHrP) at the mRNA level. Cytogenetic analysis showed the typical t(11; 14) chromosome abnormality. The novel MOLP-5 cell line together with the B407 B-LCL sister line will be useful model systems in the investigation of the biology of MM.

Novel mannose-binding rice lectin composed of some isolectins and its relation to a stress-inducible salT gene.

The novel mannose-binding rice lectin (MRL) purified by Sephadex G-50 or maltamyl Sepharose 4B affinity chromatography was not homogeneous, but the components were separated clearly by two dimensional polyacrylamide gel electrophoresis (1st; isoelectric focusing with Immobiline, 2nd; SDS-PAGE). The major spots were located at pI 4.85 and 4.74, and minor spots at pI 4.66, 4.56, and 4.44; all spots were distributed at about MW 45,000. Other faint spots were sometimes detected just below the major spots. In the western blot analysis, all the spots reacted with the monoclonal antibodies specific to MRL, which bound to MRL and inhibited the lectin activity to agglutinate rabbit erythrocytes. The proteins of the spots at pI 4.85, 4.77, 4.66, and 4.56 had lectin activity. The major proteins at pI 4.85 and 4.77 also had the common amino acid sequence at N-terminus, TLVKIGPWGGNGGSAQDISV, which is almost identical to salt and drought stress-inducible salT gene products in rice plants. High homology was also conserved in both the cDNA and the genomic clones encoding the MRL component at pI 4.85, which were selected with MRL-specific antibodies and an oligonucleotide designed from the partial amino acid sequence. All results suggest that MRL is composed of several isolectins, if not, related proteins having a common epitope and may belong to a family of stress-inducible proteins.

Serum levels of parathyroid hormone-related protein are not elevated in patients with T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare post-thymic T-cell neoplasm which shares most clinical features with adult T-cell leukemia (ATL). We measured serum level of C-terminal parathyroid hormone-related protein (C-PTHrP) in patients with T-PLL and ATL. Serum C-PTHrP levels of eight patients with T-PLL (median 36.8 pmol/l; range 27.0-50.2 pmol/l) did not differ from those of 30 human T-lymphotropic virus type I (HTLV-I)-seronegative blood donors (median 37.0 pmol/l; range 22.6-54.0 pmol/l). The C-PTHrP levels in ten ATL patients (median 69.6 pmol/l; range 42.5-899.4 pmol/l) were significantly higher than those in healthy controls (p<0.0001) or T-PLL patients (p=0.001). We suggest that the serum level of PTHrP can provide useful information for differentiating between T-PLL and ATL.

Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia.

Gene targeting studies in mice have shown that the lack of Ikaros activity leads to T-cell hyperproliferation and T-cell neoplasia, establishing the Ikaros gene as a tumor suppressor gene in mice. This prompted us to investigate whether mutations in Ikaros play a role in human hematological malignancies. Reverse transcription-PCR was used to determine the relative expression levels of Ikaros isoforms in a panel of human leukemia/lymphoma cell lines and human bone marrow samples from patients with hematological malignancies. Among the cell lines examined, only BV-173, which was derived from a chronic myelogenous leukemia (CML) patient in lymphoid blast crisis, overexpressed the dominant-negative isoform, Ik-6. In 9 of 17 samples of patients in blast crisis of CML, Ikaros activity had been reduced either by drastically reducing mRNA expression (4 of 17) or by overexpressing the dominant-negative isoform Ik-6 (5 of 17). Significantly, expression of Ikaros isoforms seemed normal in chronic phase CML patients and patients with other hematological malignancies. In some cases, overexpression of the dominant-negative Ik-6 protein was confirmed by Western blot analysis, and Southern blot analysis indicated that decreases in Ikaros activity correlated with a mutation in the Ikaros locus. In summary, these findings suggest that a reduction of Ikaros activity may be an important step in the development of blast crisis in CML and provide further evidence that mutations that alter Ikaros expression may contribute to human hematological malignancies.

Establishment and characterization of a novel ALL-L3 cell line (BALM-18): induction of apoptosis by anti-IgM and inhibition of apoptosis by bone marrow stroma cells.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-18, was established from the peripheral blood specimen of a patient with B cell ALL L3 type (ALL-L3) at diagnosis using bone marrow stroma cells (BST) as feeder cells. The primary leukemia cells did not grow without feeder cells. As with the primary leukemia cells, BALM-18 showed an immunophenotype of Burkitt's lymphoma group I [CD10+, CD20+, CD23-, CD39-, CD77+] and carried the t(8;14)(q24;q32) chromosomal abnormality which is highly associated with ALL-L3 and Burkitt's lymphoma. It also revealed a significantly low level of bcl-2 protein. Strikingly, anti-human IgM antibody did induce apoptosis in induction experiments. However, it was reversed by the addition of anti-CD40 antibody or BST cells, whereas the culture supernatant of the stroma cells did not show any effect on the inhibition of apoptosis. BALM-18 may be useful for analyzing both the mechanisms of anti-IgM induced apoptosis and signaling during the inhibition of apoptosis by CD40 or BST cells.

Establishment and characterization of "biphenotypic" acute leukemia cell lines with a variant Ph translocation t(9;22;10) (q34;q11;q22).

We established two novel PH-positive acute leukemia cell lines with "biphenotypic" feature, NALM-27 and NALM-28, with t(9;22;10)(q34;q11;q22) from a patient with biphenotypic acute leukemia(BAL). The breakpoint cluster region (bcr) of the BCR gene was found to be rearranged in these cells by Southern blot analysis using a major 3'-side bcr probe. Polymerase chain reaction (PCR) results showed differences in the pattern of expression of the abl-bcr fusion gene in comparison with the diagnosis. In the case of variant Ph translocations, reports have appeared concerning mainly chronic myelogenous leukemia (CML), but there have been few concerning acute leukemia with lymphoid feature. This study thus identifies nonrandomly involved chromosome sites which can then be targeted for detailed molecular analysis to obtain an understanding of abl-bcr fusion in the cells with lymphoid feature. In addition, chromosome band 10q22, involved in this translocation, is the site for several neoplasia. Furthermore, this site is non-randomly involved in the formation of variant Ph translocations in acute lymphoblastic leukemia (ALL). This is the first report on the t(9;22;10)(q34;q11;q22) rearrangement in NALM-27 and NALM-28 cell lines which should prove useful for understanding the translocation of molecular breaks within the bcr of the complex translocation site.

Myeloperoxidase positive acute lymphoblastic leukemia cell lines, NALM-30, NALM-31 and NALM-32, carrying Philadelphia chromosome with biphenotypic characteristics.

We established three sister cell lines, NALM-30, NALM-31 and NALM-32, with biphenotypic features carrying myeloperoxidase mRNA and protein with complex Philadelphia (Ph) chromosome, t(9;22;10)(q34;q11;q22), from a patient with Ph-positive acute leukemia in relapse. Epstein-Barr virus nuclear antigen was negative. The morphological appearance of the cell lines is that of immature lymphoid cells. Expression of myeloid- and lymphoid-associated surface membrane antigens on these cells was detected allowing for the classification of "biphenotypic" leukemia. Immunophenotypically, the established cell lines reported here fulfill the European Group for the Immunological Characterization of Leukemias (EGIL) criteria for B-lineage derivation, however, surface and cytoplasmic immunoglobulin chains were negative. Whereas TGF-beta R (CD105), MCSFR (CD115), SCFR (CD117), IL-4R/IL-13R (CD124) and IL-6R (CD126) were not expressed, the cell lines were mostly positive for IFN-gamma R (CD119), IL-7R (CD127) and FLT-3R (CD135). The NALM-30, NALM-31 and NALM-32 cell lines together with their serial sister cell lines NALM-27 and NALM-28 which were established from the same patient at diagnosis provide unprecedented opportunities for studying a multitude of biological aspects related to normal and neoplastic immature B-lymphocytes.

Interferon-gamma enhances megakaryocyte colony-stimulating activity in murine bone marrow cells.

We have demonstrated previously that interferon-gamma (IFN-gamma) accelerates platelet recovery in mice with 5-FU induced-marrow aplasia in vivo. However, the mechanism for the regulation of megakaryocyte development induced by IFN-gamma in bone marrow cells in vivo remains unknown. To further study the effects of IFN-gamma on megakaryocyte development, various steps during IFN-gamma-mediated accelerated differentiation of the megakaryocytes were investigated in serum-free cultures of murine bone marrow cells in vitro. IFN-gamma markedly induced acetylcholine esterase (AChE) activity, a marker of murine megakaryocytic cells, accompanied by increased colony formation of the megakaryocyte lineage. A prominent increase in megakaryocyte number was observed after IFN-gamma treatment. All of these effects were dependent on the presence of IL-3, and, therefore, these results suggest that IFN-gamma acts as a megakaryocyte potentiator (Meg-POT). However, IFN-gamma did not enhance megakaryocyte maturation with respect to increase in cell size. The effects of IFN-gamma on megakaryocyte maturation were similar to those observed after treatment with higher doses of IL-3 alone. Meg-POT is defined as a factor that induces megakaryocyte maturation. Since IFN-gamma enhanced IL-3-dependent megakaryocyte colony formation and proliferation rather than megakaryocyte maturation, the effects on megakaryocyte development, which were induced by IFN-gamma treatment, seem to be different from the effects of a Meg-POT. We, therefore, propose a new function for IFN-gamma as an enhancer of megakaryocyte colony-stimulating factor activity. The effect of IFN-gamma in vitro appears to correlate well with the acceleration of platelet recovery in vivo.

Purification of membrane-bound ATPase of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 and characterization as a F0F1-type enzyme.

The ATPase bound to the inner membrane of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 (Vibrio ABE-1) was extracted with Triton X-100 and purified by fractionation with polyethylene glycol, sucrose density gradient centrifugation, and DEAE-Toyopearl 650M column chromatography. The molecular masses of subunits constituting the purified ATPase were estimated as 54, 49, 33.5, 27, 23.5, 18.5, and 15 kDa by SDS-PAGE. The composition and molecular masses of the subunits of the purified ATPase were similar to those of Escherichia coli F0F1-ATPase (EF0F1). The 54-, 49-, and 18.5-kDa polypeptides of the Vibrio ABE-1 ATPase strongly cross-reacted with the antibodies against the EF0F1 alpha, beta, and b subunits, respectively. However, the Vibrio ABE-1 ATPase contained no cross-reactive polypeptide with the antibodies against A and B subunits of V-type H(+)-ATPase from mung bean tonoplasts. The ATPase activity showed two pH optimum peaks at pH 5.3 and 8.0 and was strongly inhibited by N,N'-dicyclohexyl carbodiimide (DCCD) and NaN3. It hydrolyzed ATP, GTP, and ITP at similar rates. These properties confirm that the purified ATPase is a F0F1-type. The optimum temperature for the ATP-hydrolyzing activity of the enzyme was observed at 50 degrees C, but the DCCD-sensitivity of the enzyme was markedly decreased above 30 degrees C, suggesting that the F1-moiety is released from the enzyme complex at high temperatures. This characteristic is compatible with the psychrophilic nature of Vibrio ABE-1.

IFN-gamma in combination with IL-3 accelerates platelet recovery in mice with 5-fluorouracil-induced marrow aplasia.

The effects of interferon-gamma (IFN-gamma) on platelet recovery were examined in mice with marrow aplasia induced by i.p. injection of 250 mg/kg of 5-fluorouracil (5-FU). The cytokine was administrated by microosmotic pump, with an ability to deliver a consistent intact dose of cytokine for 7 consecutive days. Administration of 250 IU/kg/day of IFN-gamma in combination with 10(3) U/kg/day of IL-3, which alone had no effect on platelet counts, diminished the nadir for platelet count and shortened the duration of thrombocytopenia. The effect was comparable to that of higher doses of IL-3 (10(5) U/kg/day). The administration of 250 IU/kg/day of IFN-gamma in combination with 10(3) U/kg/day of IL-3 also induced megakaryocyte proliferation in bone marrow cell cultures. Single administration of either 250 IU/kg/day of IFN-gamma or 10(3) U/kg/day of IL-3 had no significant effects. The effect of this combination was also comparable to that of a higher dose of IL-3 (10(5) U/kg/day). We suggest that IFN-gamma accelerates megakaryocyte development, which leads to platelet production in chemotherapy-induced marrow aplasia. The administration of IFN-gamma in combination with IL-3 might be useful for the management of marrow aplasia.

Thimerosal induces toxic reaction in non-sensitized animals.

The effects of injection of thimerosal solution on nonsensitized animals was investigated. Intrafootpad injection of thimerosal solution in nonsensitized mice resulted in a swelling response which peaked 1 h after injection and lasted for more than 24 h. Histopathological examination showed that there were severe edema and infiltration of polymorphonuclear neutrophils at the site of injection. An increased vascular permeability was observed after cutaneous injection of thimerosal solution on the back of nonsensitized rats. Since mercuric chloride and methyl mercury induced severer reactions, and thiosalicylic acid had no effect, mercury contained in thimerosal would have caused the reactions observed in this study. These results suggest that part of these hypersensitivity reactions against thimerosal observed among patients were possibly induced by the toxic effect of thimerosal. Therefore, thimerosal contained as a preservative in vaccine may augment the side-effects of the vaccination.