Irinotecan plus carboplatin for patients with carcinoma of unknown primary site.

Carcinoma of unknown primary site (CUP) is rarely encountered in clinical practice and optimal chemotherapy has not yet been established. This phase II study was conducted to evaluate the efficacy and toxicity of combined irinotecan+carboplatin therapy in chemotherapy-naive patients with CUP. Irinotecan was administered at 60 mg m(-2) as a 90-min intravenous infusion on days 1, 8 and 15. Carboplatin was administered at an area-under-the curve of 5 mg ml(-1) min as a 60-min intravenous infusion on day 1. This cycle was repeated every 28 days for up to six cycles. Forty-five patients were enrolled in the study. An intent-to-treat analysis revealed an objective response rate to the treatment of 41.9% (95% confidence interval, 27.0-57.9%). The median time to progression was 4.8 months and the median survival was 12.2 months. The 1- and 2-year survival rates were 44 and 27%, respectively. The most frequent grade 3 or more severe adverse events were leukopaenia (21%), neutropaenia (33%), anaemia (25%) and thrombocytopaenia (20%). Thus, the combination of irinotecan plus carboplatin was found to be active in patients with CUP. Therefore, the regimen may be one of the potentially available chemotherapeutic options for community standard of care in patients with a good performance status.

Mechanism of the radiosensitization induced by vinorelbine in human non-small cell lung cancer cells.

Vinorelbine (Navelbine, KW-2307), a semisynthetic vinca alkaloid, is a potent inhibitor of mitotic microtubule polymerization. The aims of this study were to demonstrate radiosensitization produced by vinorelbine in human non-small cell lung cancer (NSCLC) PC-9 cells and to elucidate the cellular mechanism of radiosensitization. A clonogenic assay demonstrated that PC-9 cells were sensitized to radiation by vinorelbine with a maximal sensitizer enhancement ratio at a 10% cell survival level of 1.35 after 24-h exposure to vinorelbine at 20 nM. After 24-h exposure to vinorelbine at 20 nM, the approximately 67% of the cells that had accumulated in the G2/M-phase were cultured in the absence of vinorelbine and then irradiated at a dose of 8 Gy. Flow cytometric analyses showed prolonged G2/M accumulation concomitant with continuous polyploidization, and induction of apoptosis was observed in the cells subjected to the combination of vinorelbine-pretreatment and radiation. Polyploidization and induction of apoptosis were confirmed by morphological examination and a DNA fragmentation assay, respectively. We concluded that vinorelbine at a minimally toxic concentration moderately sensitizes human NSCLC cells to radiation by causing accumulation of cells in the G2/M-phase of the cell cycle. Prolonged G2/M accumulation concomitant with continuous polyploidization and increased susceptibility to induction of apoptosis may be associated with the cellular mechanism of radiosensitization produced by vinorelbine.

Enhancement of in vivo antitumor activity of a novel antimitotic 1-phenylpropenone derivative, AM-132, by tumor necrosis factor-alpha or interleukin-6.

TK5048 and its derivatives, AM-132, AM-138, and AM-97, are recently developed antimitotic (AM) compounds. These 1-phenylpropenone derivatives induce cell cycle arrest at the G2 / M phase of the cell cycle. TK5048 inhibited tubulin polymerization in human lung cancer PC-14 cells in a concentration-dependent manner. In a polymerization assay using bovine brain tubulin, AM-132 and AM-138 were quite strong, AM-97 was moderately strong, and TK5048 was a relatively weak inhibitor of tubulin polymerization. A murine leukemia cell line resistant to a sulfonamide antimitotic agent, E7010, which binds to colchicine-binding sites on tubulin, was cross-resistant to the in vitro growth-inhibitory effect of AM compounds. Inhibition of tubulin polymerization is therefore one of the mechanisms of action of these AM compounds against tumor cells. To profile the antitumor effect of AM compounds, the in vivo antitumor effect of AM-132 was evaluated against cytokine-secreting Lewis lung carcinoma (LLC). Tumor-bearing mice were treated with intravenous AM-132 using three different treatment schedules. LLC tumors expressing tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF), or interleukin (IL)-6 were very sensitive to AM-132. In particular, LLC tumors expressing IL-6 were markedly reduced by AM-132 treatment, and showed coloring of the tumor surface and unusual hemorrhagic necrosis. These results suggest a combined effect of AM-132 and cytokines on the blood supply to tumors.

Prolonged bone marrow failure with monosomy 7 after engraftment failure following bone marrow transplantation.

A patient with acute myelogenous leukemia developed prolonged bone marrow failure along with the monosomy 7 chromosome abnormality. The patient had undergone bone marrow transplantation with CD34+ selection following induction failure. However, she then suffered engraftment failure and long-term pancytopenia. Her white blood cell count gradually increased with supportive therapy including granulocyte colony-stimulating factor (G-CSF), and chromosomal analysis of bone marrow cells revealed an abnormal karyotype. Thirty months after the bone marrow transplantation we observed monosomy 7 together with the existing chromosomal abnormality in the patient's bone marrow cells. It has been reported that some patients with idiopathic and posthepatitis aplastic anemia develop clonal disorders such as myelodysplastic syndrome/acute myelogenous leukemia with monosomy 7. The findings in our case suggest that the appearance of monosomy 7 in patients with aplastic anemia may be caused by prolonged low-level hematopoiesis, with or without G-CSF stimulation.

In vivo effects of apoptosis in asthma examined by a murine model.

BACKGROUND: One of the characteristic features of bronchial asthma is the accumulation of various inflammatory cells, predominantly eosinophils, at the subepithelial region beneath the basement membrane of the airway. Apoptosis is a form of physiological cell death, through which the cellular contents including biologically active substances are kept in the cell membrane and are removed without their harmful effects. So, attempts were made to clarify whether the induction of apoptosis is beneficial in asthma by using a murine model with ovalbumin (OA) as responsible allergen. METHODS: A/J mice, which are genetically predisposed to be hyperresponsive to acetylcholine, were immunized with OA and alum, accompanied by OA inhalation for 2 weeks, during which some of the mice were also treated with either anti-Fas monoclonal antibody or sham control hamster IgG intranasally. Airway responsiveness to acetylcholine was then analyzed by measuring airway resistance with a body plethysmograph box. Apoptosis was assessed by propidium iodide and TUNEL staining. RESULTS: Inhalation of OA increased both airway responsiveness to acetylcholine and the number of cells, mostly eosinophils, infiltrated into the airway. Administration of anti-Fas antibody induced apoptosis in the infiltrated eosinophils and abolished augmentation of airway hyperresponsiveness caused by OA inhalation. CONCLUSION: Induction of apoptosis in proinflammatory cells including eosinophils at the airway may have a beneficial effect on suppressing airway hyperresponsiveness.

[Anaplastic thyroid carcinoma with lung metastasis producing CA 19-9 and GM-CSF].

A 68-year-old Japanese woman was admitted to our hospital because of hoarseness, dysphagia and a mass on the right side of her neck. Chest radiographs showed multiple nodular shadows in both lung fields. Detailed investigations resulted in a diagnosis of multiple lung metastasis of anaplastic thyroid carcinoma transformed from papillary adenocarcinoma. Both serum CA 19-9 and GM-CSF levels were elevated, to 70.5 U/ml (normal range: 0-37 U/ml) and 343.4 pg/ml (normal range: 0-8 pg/ml), respectively. Immunostaining disclosed that the primary and metastatic tumors were positive for CA 19-9, but not for GM-CSF antigens. Serum levels of these two parameters slowly decreased after chemo-radiotherapy, suggesting that the tumor may have produced GM-CSF as well as CA 19-9. Recent studies have indicated that the prognosis is poor for non-small cell lung cancers that produce G-CSF or CA 19-9. To our knowledge, this is the first case report of anaplastic thyroid carcinoma characterized by high serum levels of both CA 19-9 and GM-CSF, with metastasis to the lung and other organs.

Induction of apoptosis in bronchial eosinophils: beneficial or harmful?

BACKGROUND: Prominent eosinophil infiltration takes place in asthmatic bronchi, and damages bronchial epithelial cells. AIM: This study was designed to investigate whether induction of apoptosis in infiltrated cells in the airways is beneficial or harmful. METHODS: A/J mice, which are genetically predisposed to be hyperresponsive to acetylcholine, were immunized with ovalbumin (OA) and alum. Thereafter, they were subjected to a 2-week regimen of OA inhalation, during which they were also administered either hamster anti-mouse Fas monoclonal antibody or hamster IgG (sham control) intranasally. Pulmonary function was then analyzed using whole-body plethysmography. RESULTS: Inhalation of OA increased both airway responsiveness to acetylcholine and infiltration of eosinophils. Administration of anti-Fas antibody induced apoptosis in the infiltrating eosinophils and abolished the increase in airway responsiveness to acetylcholine. CONCLUSION: Induction of apoptosis in eosinophils infiltrating asthmatic bronchi has a beneficial effect on airway hyperresponsiveness.

Ectopic p16(ink4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in human non-small-cell-lung-cancer cells.

A tumor-suppressor gene, p16(INK4), which is deleted or mutated in tumors, regulates cell-cycle progression through a G(1)-S restriction point by inhibiting CDK4(CDK6)/cyclin-D-mediated phosphorylation of pRb. We have found that ectopic p16(INK4) expression increased cellular sensitivity of human non-small-cell-lung-cancer (NSCLC) A549 cells to a selective growth-inhibitory effect induced by the topoisomerase-I inhibitor 11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) in vitro. In this study, we observed enhanced apoptosis characterized by DNA fragmentation in A549 cells transfected with p16(INK4) cDNA (A549/p16-1) and treated with CPT-11. This apoptosis was suppressed by the inhibitor of interleukin-1beta-converting enzyme (ICE/caspase-1) or ICE-like proteases, Z-Asp-CH2-DCB, as determined by DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase, a natural substrate for CPP32/caspase-3. In A549/p16-1 cells, cytosolic peptidase activities that cleaved Z-DEVD-7-amino-4-trifluoromethylcoumarin increased during CPT-11-induced apoptosis and were suppressed by a highly specific caspase-3 and caspase-3-like inhibitor, Z-DEVD-fluoromethylketone. These findings indicate that p16(INK) is positively involved in the activation pathway of the caspase-3 induced by CPT-11. The increased delay in S-phase progression and subsequent induction of apoptosis were observed in CPT-11-treated A549/p16-1 cells on the basis of DNA histograms. Specific down-regulation of the cyclin-A protein level in A549/p16-1 cells was observed after CPT-11-treatment, whereas cyclin B, cdk2, and cdc2 protein levels were unaffected. These results suggest that ectopic p16(INK4) expression inappropriately decreases cyclin A and thereby terminates CPT-11-induced G(2)/M accumulation, which is followed by increased apoptosis in p16(INK4)-expressing A549 cells.

A diffuse alveolar hemorrhage in a human T-lymphotropic virus type I carrier with acute cerebellar ataxia and interstitial pneumonitis: an autopsy case report.

A 76-year-old HTLV-I-positive male with acute cerebellar ataxia was suffering from dyspnea on exertion. Chest CT suggested interstitial pneumonitis. Methylprednisolone pulse therapy improved his symptoms and chest CT findings. Twelve months after discharge, when the prednisolone dose was tapered to 5 mg every other day, his lung lesion recurred. The lesion responded initially to steroid therapy. However, hypoxemia intractable to steroid pulse therapy developed and the patient died of respiratory failure. The autopsy revealed diffuse alveolar hemorrhage with no finding of vasculitis. This is the first case report of diffuse alveolar hemorrhage in an HTLV-I carrier.

Neurological complications after stem cell transplantation in childhood.

We analyzed the incidence of neurological complications in 77 patients receiving stem cell transplantation (SCT), and 12 patients (15.8%) had the following symptoms: convulsions, intracranial hemorrhage, and leukoencephalopathy. Although statistically not significant, neurological complications were seen more frequently in patients after allogeneic transplantation, and in those with acute graft-versus-host disease (GVHD) exceeding grade II. The most significant risk factor for neurological complications was identified as unrelated donor allogenic transplantation (P = 0.016). Complications were categorized into three groups, based on time of onset and symptoms: (1) convulsions during the conditioning period, (2) intracranial hemorrhage during the period of granulocyte recovery, and (3) leukoencephalopathy at around 2 months after SCT. We propose awareness of the risks of neurological complications in each period after SCT so that immediate and effective treatment of patients can be instigated.

[Adverse effects of anti-thymocyte globulin/anti-lymphocyte globulin therapy].

This single-centre study evaluated the adverse effects of anti-thymocyte globulin (ATG) and anti-lymphocyte globulin (ALG) as used for the treatment of aplastic anemia and/or for conditioning regimens prior to stem cell transplantation. ATG/ALG was given to 29 patients a total of 37 times. The incidence of adverse effects was 62.1% (23/37), and fever was the most frequent adverse effect. Therapy was discontinued in only 4 patients (10.8%) due to severe adverse effects. Adverse effects occurred more frequently with ATG (rabbit-derived) than with ALG (horse-derived). Seven patients underwent 2 or 3 cycles of ATG/ALG therapy, for a combined total of 8 times; 6 of those patients (75% (6/8)) experienced adverse effects. Shorter intervals between repeated cycles of therapy appeared to heighten the risk of adverse reactions.

[Vasculo-Behcet's disease with fatal massive hemoptysis].

A 39-year-old man was admitted to our hospital because of hemoptysis. A chest X-ray film on admission showed a patchy shadow in the left lower lung field. Computed tomography revealed nodular opacities in the left pulmonary artery. The patient had history of oral ulcers, erythema nodosum, pustular lesions, and genital ulcers. Furthermore, the needle reaction was positive. Our diagnosis was an incomplete type of Behcet's disease. A radionuclide-venography and lung-perfusion study disclosed deep-vein thrombosis. Combined therapy with prednisolone, colchicine, and indomethacin farnesil was initiated, but the patient died of massive hemoptysis. Pathological examination revealed a ruptured aneurysm in the bronchus segmentalis apacalis and thrombotic angitis in the inferior vena cava. Behcet's disease is rarely a cause of hemoptysis. However, the prevalence of hemoptysis due to pulmonary vasculitis in patients with Behcet's disease has been reported to be 5 to 10% which is not so rare. Because of the poor prognosis, we want to emphasize Behcet's disease as a cause of hemoptysis.

Enhancement of cisplatin sensitivity in high mobility group 2 cDNA-transfected human lung cancer cells.

To elucidate the role of high mobility group 2 protein (HMG2) in cis-diamminedichloroplatinum (II) (cisplatin, CDDP) sensitivity, we constructed a human HMG2-transfected human non-small cell lung cancer cell line, PC-14/HMG2. The HMG2 mRNA expression level was approximately twice those of parental PC-14 and mock-transfected PC-14/CMV. Gel mobility shift assay revealed a CDDP-treated DNA-protein complex in the nuclear extract of PC-14/HMG2, which was not found in the extracts of PC-14 and PC-14/CMV. This complex formation was subject to competition by CDDP-treated non-specific salmon sperm DNA, indicating that ectopic HMG2 recognizes CDDP-damaged DNA. PC-14/HMG2 showed more than 3-fold higher sensitivity to CDDP than PC-14 and PC-14/CMV. The intracellular platinum content of PC-14/HMG2 after exposure to 300 microM CDDP was 1.1 and 1.5 times that of PC-14 and PC-14/CMV, respectively. Cellular glutathione levels were not different in these cell lines. Repair of DNA interstrand cross-links determined by alkaline elution assay was decreased in PC-14/HMG2. These results suggest that HMG2 may enhance the CDDP sensitivity of cells by inhibiting repair of the DNA lesion induced by CDDP.

Electroencephalogram abnormality and high-dose busulfan in conditioning regimens for stem cell transplantation.

High-dose busulfan (BU) is widely used in combined chemotherapy before allogeneic or autologous bone marrow transplantation. Convulsions are reported as a side-effect of high-dose BU. We recorded electroencephalograms (EEGs) before and on the third day of BU administration in 22 patients. Abnormal EEGs were observed on the third day in 13 cases (59%). These patients were older (P < 0.05) and had had larger doses of BU (P < 0.025) than the nine patients with normal EEGs. Convulsions occurred in two of the 22 patients, one of whom was receiving prophylaxis with phenytoin. Gamma aminobutyric acid (GABA), a natural mediator of defense against epileptic activity, concentrations in cerebrospinal fluid measured before and after administration of BU showed no definite changes.

The C-terminal domain of p53 catalyzes DNA-renaturation and strand exchange toward annealing between intact ssDNAs and toward eliminating damaged ssDNA from duplex formation through preferential recognition of damaged DNA by a duocarmycin.

The C-terminal domain of p53 may bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules, suggesting a possible direct role for p53 in DNA repair (Proc. Natl. Acad. Sci. USA, 92, 9455-9459, 1995). We found that DU-86, a duocarmycin derivative which alkylates DNA, bound ssDNA and enhanced the DNA binding activity of the p53 C-terminus. DU-86 weakened p53-mediated catalysis of complementary ssDNA renaturation. p53 C-terminus catalyzed DNA strand transfer toward annealing between intact ssDNAs and toward eliminating DU-86-damaged ssDNA from duplex formation. These results suggest that p53, via the C-terminal domain, may play a direct role in DNA repair by preferential recognization and elimination of damaged DNA.

Circumvention of glutathione-mediated mitomycin C resistance by a novel mitomycin C analogue, KW-2149.

A novel antitumor antibiotic 7-N-[2-[[2-(gamma-L-glutamylamino)ethyl]dithio]ethyl] mitomycin C (KW-2149), an analogue of mitomycin C (MMC), is activated by thiol molecules, such as glutathione (GSH). To clarify the relationship between cellular GSH levels and the cytotoxicity of KW-2149, a murine fibroblast cell line (NIH/3T3) was transfected with human gamma-glutamylcysteine synthetase (gamma-GCS) cDNA, which codes a rate-limiting enzyme of GSH synthesis. Transfected cells (3T3/GCS) displayed increased gamma-GCS mRNA levels, gamma-GCS activity and GSH content, compared with NIH/3T3 cells. 3T3/GCS cells exhibited a 4.4-fold resistance to MMC, but not to KW-2149 (x 0.69), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, suggesting that the increased cellular GSH levels did not affect the growth-inhibitory effect of KW-2149. KW-2149 exerted a greater growth-inhibitory effect than MMC on cisplatin- and doxorubicin-resistant cells with cross-resistance to MMC. KW-2149 exhibited a greater growth inhibitory effect than MMC not only on cells with GSH-mediated MMC resistance but also on cells with acquired resistance. We thus conclude that KW-2149 might be a clinically useful drug.

Interferon-gamma-inducing factor gene transfection into Lewis lung carcinoma cells reduces tumorigenicity in vivo.

To investigate the immunoregulatory effect of murine interferon-gamma-inducing factor (mIGIF), we transfected Lewis lung carcinoma (LLC) cells with a mammalian expression vector containing the mIGIF complementary DNA. The culture medium of the transfectant cells stimulated interferon-gamma (IFN-gamma) production by spleen cells in vitro in the presence of anti-CD3 antibody and markedly potentiated the effect of interleukin-12 (IL-12) on IFN-gamma production by spleen cells. mIGIF transfectant cells showed reduction of tumorigenicity and induction of an in vivo immuno-protective effect against the parental LLC cells. To examine the combined effect of systemic administration of recombinant IL-12 (rIL-12) and local mIGIF on the tumorigenicity, mice were challenged with LLC or transfectant cells on day 0, and the tumor-bearing mice were injected with 50 ng of rIL-12 intraperitoneally from day 7 to 11. Systemic rIL-12 showed an anti-tumor effect. However, mIGIF gene expression did not potentiate this effect of systemic rIL-12 in vivo.

Identification of cis-acting DNA elements of the human gamma-glutamylcysteine synthetase heavy subunit gene.

Transcriptional activity of the 5'-flanking sequence of the human gamma-glutamylcysteine synthetase heavy subunit (gamma-GCSh) gene was investigated in COS7 cells transfected with hGH reporter constructs having successively deleted 5'-flanking sequence of the gamma-GCSh gene. Transcriptional activity was determined by the amounts of hGH secreted from the reporter constructs. Deletion of the sequence from -1,413 to -664 or -315 base pairs (bp) increased transcriptional activity from 100 to 138 or 136%. Further deletion from -315 to -241 bp, which contained an AP1 site, decreased transcriptional activity to 87%. Mutations introduced into the AP1 decreased transcriptional activity from 136 to 105%. These findings suggested that the AP1 increased transcriptional activity. When the sequence from -241 to -192 bp was deleted, transcriptional activity was restored from 87 to 128%. When this sequence was linked to the thymidine kinase promoter, it also decreased transcriptional activity by 38%. Deletion from -192 to -149, -116, or -108 bp did not significantly alter transcriptional activity. Further deletion of the GC-rich sequences from -108 to -70 and -28 bp dramatically decreased transcriptional activity from 135 to 87 and 34%, respectively. These findings indicate that multiple DNA elements, especially those in the proximal GC-rich sequences, are involved in the regulation of transcriptional activity of the gamma-GCSh gene.

Effect of glutathione depletion on cisplatin resistance in cancer cells transfected with the gamma-glutamylcysteine synthetase gene.

Overexpression of the human gamma-glutamylcysteine (gamma-GCS) gene resulted in cisplatin resistance with an increased glutathione (GSH) content, increased ATP-dependent glutathione S-conjugate export pump (GS-X pump) activity and decreased platinum accumulation in human lung cancer cells transfected with a gamma-GCS cDNA expression vector, as we previously reported. In this study, we examined the effects of buthionine sulfoximine (BSO), a specific inhibitor of gamma-GCS, to determine whether GSH depletion alters cisplatin resistance in a gamma-GCS-transfected cell line, SBC-3/GCS. In the presence of 10 microM BSO for 4 days, SBC-3/GCS still showed resistance to cisplatin, although it was partially reversed. Under these conditions, GS-X pump activity remained up-regulated in spite of low GSH content, and the platinum content was decreased. These data suggest that the GS-X pump itself influences cisplatin resistance, as well as cellular GSH content.

In vitro and in vivo modulation by rhizoxin of non-P-glycoprotein-mediated vindesine resistance.

Rhizoxin is an antineoplastic drug that inhibits tubulin polymerization. In this study, we demonstrated that rhizoxin was approximately twice as active in vitro against a human small-cell lung cancer cell line with non-P-glycoprotein-mediated resistance to vindesine, H69/VDS, as against its parental line, H69. Tubulin polymerization in H69/VDS, demonstrated by Western blot analysis, was inhibited markedly by rhizoxin compared with that in H69, in a concentration-dependent manner. A drug-accumulation study showed that the intracellular rhizoxin level in H69/VDS was 15% lower than that in H69, whereas efflux from H69/VDS was enhanced slightly. These results indicate that enhanced inhibition of tubulin polymerization rather than increased intracellular drug concentration accounted for the higher sensitivity of H69/VDS to rhizoxin. In an experiment using mice with severe combined immunodeficiency and inoculated subcutaneously with H69/VDS, in vivo tumor growth was reduced markedly by three intermittent intraperitoneal doses of rhizoxin compared with that in mice inoculated with H69. Three weeks after the last rhizoxin dose, the relative treated/untreated tumor volumes were 0.29 for H69, but only 0.06 for H69/VDS, indicating that H69/VDS regrowth was minimal even after a 3-week treatment-free period. In conclusion, rhizoxin conquers vindesine resistance of a human small-cell lung cancer cell line in vitro and in vivo.