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Disaster preparation is an important issue for patients with amyotrophic lateral sclerosis (ALS). However, to the best of our knowledge, no studies have investigated disaster preparedness among patients with ALS. In this study, we aimed to investigate disaster preparation in patients with ALS and their caregivers, including their families, in Japan. We conducted a nationwide webinar in September 2022 titled "ALS Café" and distributed a self-report questionnaire to participants with questions about awareness of disaster preparedness, social countermeasures, stockpiles, and electricity demand. Forty-eight patients with ALS (27 male; average age 60.0 ± 9.3 years) and 23 caregivers (8 male; 55.7 ± 9.9 years) responded. The median revised ALS Functional Rating Scale score was 30.5, and 25% of the patients with ALS were on a ventilator. More than 70% of the respondents answered that they were not prepared for disasters, increasing to 89% in patients not using ventilators. In the event of their phones being down, 86% of the respondents had no plans for alternative means of communication. <30% of the respondents, including ventilator users, had secured human resources for transportation. Twenty-five percent of the respondents did not stockpile food and beverages, and 12% of the ventilator users had no government-recommended ventilator preparation equipment. Thus, although patients with ALS and their families with ventilators have a high awareness of disaster preparedness, their awareness remains insufficient. Furthermore, patients with ALS and their families without ventilators have a low awareness of disaster preparedness. Therefore, better education regarding disaster preparedness is necessary for these groups., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

TAFRO syndrome is a systemic inflammatory disease of unknown aetiology. It is characterised by thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly. Herein, we report the case of a 60-year-old male with TAFRO syndrome. A few weeks after the patient developed an intermittent fever, he presented to our hospital with diarrhoea, abdominal distension, and whole-body oedema (face, extremities, and abdomen). Autoantibody and lip biopsy findings supported the diagnosis of primary Sjögren's syndrome. High-dose steroids and tocilizumab were used to treat his refractory thrombocytopenia and ascites. However, systemic inflammation and renal dysfunction did not improve, resulting in temporary haemodialysis. Eventually, combined B-cell immunomodulation therapy with rituximab and belimumab ameliorated the patient's symptoms. About 16 weeks after discharge, the overall condition of the patient had improved. The TAFRO syndrome may be a severe manifestation of primary Sjögren's syndrome. Considering the immunological context, combined B-cell immunomodulation therapy provides new insights into improving this life-threatening disease and enables rapid steroid tapering., (© Japan College of Rheumatology 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Poly (ADP-ribose) glycohydrolase (PARG) is the main enzyme responsible for catabolism of poly (ADP-ribose) (PAR), synthesized by PARP. PARG dysfunction sensitizes certain cancer cells to alkylating agents and cisplatin by perturbing the DNA damage response. The gene mutations that sensitize cancer cells to PARG dysfunction-induced death remain to be identified. Here, we performed a comprehensive analysis of synthetic lethal genes using inducible PARG knockdown cells and identified dual specificity phosphatase 22 (DUSP22) as a novel synthetic lethal gene related to PARG dysfunction. DUSP22 is considered a tumor suppressor and its mutation has been frequently reported in lung, colon, and other tumors. In the absence of DNA damage, dual depletion of PARG and DUSP22 in HeLa and lung cancer A549 cells reduced survival compared with single-knockdown counterparts. Dual depletion of PARG and DUSP22 increased the apoptotic sub-G 1 fraction and upregulated PUMA in lung cancer A549, PC14, and SBC5 cells, and inhibited the PI3K/AKT/mTOR pathway in A549 cells, suggesting that dual depletion of PARG and DUSP22 induced apoptosis by upregulating PUMA and suppressing the PI3K/AKT/mTOR pathway. Consistently, the growth of tumors derived from double knockdown A549 cells was slower compared with those derived from control siRNA-transfected cells. Taken together, these results indicate that DUSP22 deficiency exerts a synthetic lethal effect when combined with PARG dysfunction, suggesting that DUSP22 dysfunction could be a useful biomarker for cancer therapy using PARG inhibitors. SIGNIFICANCE: This study identified DUSP22 as a novel synthetic lethal gene under the condition of PARG dysfunction and elucidated the mechanism of synthetic lethality in lung cancer cells., (©2019 American Association for Cancer Research.)

Despite the development of new drugs for multiple myeloma (MM), the prognosis of MM patients with high-risk cytogenetic abnormalities such as t (4; 14) and del17p remains poor. We reported that a novel phenylphthalimide derivative, TC11, induced apoptosis of MM cells in vitro and in vivo, and TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1). However, TC11 showed low water solubility and poor pharmacokinetic properties. Here we synthesized a water-soluble TC11-derivative, PEG(E)-TC11, in which HOEtO-TC11 is pegylated with PEG through an ester bond, and we examined its anti-myeloma activity. We observed that PEG(E)-TC11 and its hydrolyzed product, HOEtO-TC11, induced G2/M arrest and the apoptosis of MM cells. Intraperitoneal administration of PEG(E)-TC11 to xenografted mice revealed improved pharmacokinetic properties and significantly delayed tumor growth. TC11 and its derivatives did not bind to cereblon (CRBN), which is a responsible molecule for thalidomide-induced teratogenicity. These results suggest that PEG(E)-TC11 is a good candidate drug for treating high-risk MM., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Poly(ADP-ribose) polymerases (PARPs) family proteins catalyze poly(ADP-ribosylation) (PARylation) by conjugating ADP-ribose residues repeatedly on amino acid residues using nicotinamide adenine dinucleotide as a substrate. The inhibitors of PARP widely block DNA repair processes and are currently examined in clinical trials of cancer therapy. Poly(ADP-ribose) glycohydrolase (PARG) is the main nuclear enzyme, which digests poly(ADP-ribose) into ADP-ribose. PARG inhibitor could also be considered as a chemotherapeutic agent for cancer, because of its involvement in DNA repair. Various PARG inhibitors with IC50 value of micromolar to submicromolar range have been reported. However, for most of these chemicals, the specificity of inhibition has not been fully evaluated. PARG functional inhibition models in various organisms have been developed. Here, inducible PARG knockdown system was developed in HeLa cells and the cell line will be useful for identifying the synthetic lethal genes or affecting genes for PARG inhibitor treatment and also for functional elucidation of PARP superfamily molecules.

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

Rationale and Objectives: It is well-known that amphetamine induces increased locomotor activity in rodents. We previously found that intracerebroventricular (i.c.v.) administration of p-hydroxyamphetamine (p-OHA), an amphetamine metabolite, increases synaptic dopamine (DA) levels in the striatum. In the present study, we investigated the effect of p-OHA on locomotor activity in rodents., Results: In mice, i.c.v. administration of p-OHA significantly increased locomotor activity in a dose-dependent manner. p-Hydroxynorephedrine, another amphetamine metabolite, did not increase locomotor activity. This effect of p-OHA was inhibited by pretreatment with nomifensine, a dopamine-uptake inhibitor, but not by fluoxetine, a serotonin-uptake inhibitor, or diethyldithiocarbamate, a dopamine-beta-hydroxylase inhibitor. Furthermore, we tested the effects of microinjections of p-OHA into the rat nucleus accumbens (NAc) on locomotor activity. Local infusion of p-OHA into the NAc significantly increased locomotor activity. As in mice, the increased locomotor activity induced by p-OHA microinjection into the NAc in rats was inhibited by nomifensine., Conclusions: These data suggest that dopaminergic systems in the NAc may play important roles in p-OHA-induced locomotor activity in rodents.

Background: Although many reports on the treatment of hepatocellular carcinoma (HCC) by microwave coagulation have been published recently, none have incorporated data for the long-term therapeutic efficacy of laparoscopic microwave coagulation (LMC). In the current study, the efficacy of LMC was assessed., Methods: The authors performed LMC under local anesthesia in 69 previously untreated patients with solitary HCCs < or = 4.0 cm in greatest dimension. The maximum diameter for the tumors averaged 22.6 +/- 7.4 mm. Long-time survival rate was evaluated according to the size and histologic grade of the tumor., Results: The 5-year overall cumulative survival rate for the 69 patients was 63.9%. The 5-year overall survival rate for patients with well differentiated HCC was 78.9%, whereas patients with moderately or poorly differentiated HCC had a 5-year overall survival rate of 38.9%. The 5-year cumulative survival rate for patients with HCCs < or = 2.0 cm in diameter was 76.0%, and 56.3% for patients with HCCs >2.0 cm. Twelve patients (17.4%) showed local tumor recurrence during the follow-up period. Local tumor recurrence was observed in 6 of 21 patients with moderately or poorly differentiated HCCs (28.6%) and in 6 of 40 patients with well differentiated HCCs (15.0%). The 3-year cancer-free survival rate for patients with well differentiated HCC was 44.4%, whereas it was 12.2% for patients with moderately or poorly differentiated HCC., Conclusions: A major factor that influenced outcome in LMC was tumor cell differentiation. LMC procedures were best suited for treatment of well differentiated HCC., (2005 American Cancer Society.)

Background: The use of a new end-fire type laparoscopic US probe with a forward-viewing convex-array transducer allows the caudate lobe of the liver to be accessed. This study evaluated the preliminary results of treatment of hepatocellular carcinoma in the caudate lobe by using this new instrument., Methods: Three patients with hepatocellular carcinoma in the caudate lobe were selected. A laparoscopic US probe, with a forward-viewing convex-array transducer at the tip and a guide groove for puncture on the back, was used to monitor the position of the radiofrequency ablation needle during the treatment., Results: Ablation was performed without complication in all cases. Complete necrosis of the tumor was confirmed by postoperative CT. At a mean follow-up of 30.3 months, no local recurrence was observed in any patient., Conclusions: Radiofrequency ablation of hepatocellular carcinoma in the caudate lobe of the liver by using a new laparoscopic US probe with a forward-viewing convex-array transducer at the tip was safe and effective.

It is well known that head-twitch response (HTR) in mice represents hallucinations, since administration of lysergic acid diethylamide (LSD) produces hallucinations in humans, and the HTR in mice is induced by administration of LSD as a hallucinogen. The HTR is produced by excitation of 5-hydroxytryptamine (5-HT)2A receptors. In this paper, we review the mechanisms of HTR induced by various drugs such as 5-HT precursor, 5-HT receptor agonist, 5-HT releaser, hallucinogenic compounds, benzodiazepins and cannabinoid. The response induced by HTR-inducers is significantly enhanced by combined treatment with a non-selective form of monoamine oxidase (MAO) inhibitor. Thus, the relationship between MAO activity and HTR caused by these drugs (especially, alpha-methylated analogous compounds which 5-fluoro-alpha-methyltryptamine, 6-fluoro-alpha-methyltryptamine and p-hydroxyamphetamine) is presented in detail.

Background: It is technically difficult to puncture deep-seated hepatic tumors by conventional laparoscopic ultrasonography with a linear-array probe. We have developed a laparoscopic ultrasonography system with a convex-array probe., Methods: The laparoscopic system used had a fixed forward-viewing convex-array transducer, and a guide groove for puncture was added to the back of the unit. These characteristics enabled us to continuously monitor the position of the needle tip on the ultrasonographic image immediately after puncturing on the liver surface. We attempted tumor puncture in 11 patients with hepatocellular carcinoma under a new probe guidance., Results: The mean puncturing distance up to the tumors was 38.7 mm. All punctures were successful on the first pass and the tumors were treated with radiofrequency ablation., Conclusion: Using this new equipment, puncturing hepatic tumors for treatment is relatively easy, irrespective of the position of the tumor.

Background: Recurrent small bowel obstruction caused by postoperative adhesions has traditionally been treated by conventional laparotomy, but laparoscopic management of acute small bowel obstruction has been reported. The aim of this study was to assess the long-term efficacy and clinical outcome of laparoscopic adhesiolysis for recurrent small bowel obstruction., Methods: After conservative treatment, elective laparoscopic treatment was attempted in 17 patients hospitalized for recurrent small bowel obstruction after abdominal or pelvic surgery., Results: Postoperative adhesions were identified laparoscopically in all patients. Laparoscopic treatment was possible in 14 patients (82.4%). Conversion to laparotomy was required for 3 patients (17.6%) because of intestinal perforation (n = 1) or a convoluted mass of adherent bowel (n = 2). Long-term follow-up was possible in 16 patients. Two recurrences of small bowel obstructions were noted over a mean follow-up period of 61.7 months., Conclusions: Laparoscopic adhesiolysis is a safe and effective treatment for recurrent small bowel obstruction. Conversion to laparotomy should be considered in patients with dense adhesions.

Intraperitoneal injection of benzodiazepine receptor agonists (estazolam, zopiclone, triazolam: 0.03-0.24 mmol/kg) induces the head twitch response (HTR). The present study was undertaken to examine the possible participation of the serotonergic system in the mechanism of head twitches induced by benzodiazepine receptor agonists (BZ-RAs). The HTR induced by BZ-RAs was suppressed by pretreatment with ketanserine (1 mg/kg, i.p.), a selective 5-HT(2) receptor antagonist. Pretreatment with fluoxetine (10 mg/kg, i.p.), a 5-HT reuptake inhibitor, and 8-hydroxy-2-(di-n-propylamino)tetralin, a 5-HT(1A) receptor agonist, also suppressed the HTR induced by BZ-RAs. These results suggest that the HTR induced by BZ-RAs may be the result of an activation of postsynaptic 5-HT(2) receptors, probably due to direct action., (Copyright 2001 S. Karger AG, Basel)

Background: Detection of small hepatocellular carcinomas has become possible with improvements in various diagnostic imaging techniques. However, intraoperative US can detect lesions not visualized by any preoperative imaging study in which case it is difficult to determine whether the lesion is a hepatocellular carcinoma., Methods: Nodular lesions detected by laparoscopic US in 186 patients with hepatocellular carcinoma were examined and we evaluated the diagnostic ability of laparoscopic US to detect multicentric hepatocellular carcinoma., Results: One hundred thirty-four new nodular lesions were detected by laparoscopic US in 64 (34.4%) of 186 patients. Aspiration biopsy under laparoscopic US guidance was performed on the 134 nodules, and 28 nodules in 23 (12.4%) of the 186 patients were histologically diagnosed as hepatocellular carcinoma. Of these 23 patients, 18 had been diagnosed with solitary hepatocellular carcinoma before laparoscopic US. One hundred six of the newly detected lesions were initially diagnosed as noncarcinomatous nodules, but the diagnosis of 10 of these lesions was changed to hepatocellular carcinoma during follow-up that was as long as 96 months., Conclusions: Laparoscopic US is useful in the initial diagnosis of hepatocellular carcinoma and impacts treatment selection by more accurately defining the presence of multicentric hepatocellular carcinomas.

Background: Most patients with hepatocellular carcinoma have underlying cirrhosis, and this impairment of liver function makes hepatectomy difficult, prompting the use of other modalities such as transcatheter arterial embolization and percutaneous ethanol injection., Methods: Laparoscopic ethanol injection was performed in 48 previously untreated patients with hepatocellular carcinoma smaller than 2 cm in diameter. Long-term survival was evaluated., Results: In 12 patients, hepatocellular carcinoma was not detected by trans-cutaneous ultrasonography but could be demonstrated by laparoscopic ultrasonography. Laparoscopic ethanol injection did not cause serious complications in any patient. The mean hospital stay after ethanol injection was 8.6 days (4 to 15 days). The cumulative survival rate was 86.7% at 3 years and 60.0% at 5 years. According to the Child-Pugh classification, the cumulative survival rate at 5 years was 87.9% for class A, 65.7% for class B, and 28.6% for class C., Conclusions: The long-term prognosis for patients with small hepatocellular carcinoma treated solely by laparoscopic ethanol injection is satisfactory but still dependent on underlying liver function.

Differentiation therapy in patients with acute promyelocytic leukemia (APL) using all-trans-retinoic acid (ATRA) has now been established as an effective strategy for the treatment of leukemia. However, the clinical response of patients with leukemias other than APL to differentiation inducers is limited, often with inconsistent outcome. Recently, numerous new differentiation inducers for various leukemia cells have been developed, some of which have had therapeutic effects on various leukemias in preclinical and clinical trials. Additionally, molecular control mechanisms of differentiation, and apoptosis of various leukemia cells by differentiation inducers have been studied extensively. These studies suggest that problems underlying the current discrepancy between preclinical and clinical therapeutic efficacy of leukemias can be overcome by these basic studies together with multidisciplinary studies on the therapy of leukemias in combination with differentiation therapy and other conventional therapies.

Laparoscopic microwave coagulation (LMC) for hepatocellular carcinomas (HCCs) was performed on 26 HCCs in 17 patients. Contrast-enhanced CT (CECT) and MR images (T1-weighted imaging [T1WI], T2WI, gadolinium-diethylenetriamine pentaacetic acid [Gd-DTPA] T1WI) were obtained to determine changes over time. The irradiated center exhibited low to moderate intensity with surrounded high intensity (HI) on T2WI and Gd-DTPA T1WI. On T1WI, lesions showed four patterns of intensity: uniform HI (30.8%), arcuate HI (26.9%), mainly low with spot HI (30.8%), and isointensity to hypointensity (11.5%). Follow-up imaging at more than 170 days revealed isointensity to hypointensity on T1WI (96.2%) and reduced HI on T2-weighted imaging (T2WI) and Gd-DTPA T1WI. All lesions became less conspicuous and were reduced in volume. HCC shows time-related changes in signals and size after LMC. Identifying the irradiated lesion is necessary to estimate the adequacy of treatment by comparison with the pretherapeutic image.

Plasma beta-carotene, alpha-tocopherol and retinol were measured in 15 female and 5 male children with insulin-dependent diabetes mellitus (IDDM), and the correlations with plasma hemoglobin A1c (HbA1c) and fructosamine were analyzed. Twelve female and 8 male children served as age-matched controls. The plasma beta-carotene and alpha-tocopherol levels of the IDDM children were significantly higher than those of the control children, but there were no differences in plasma retinol or total lipid levels. The plasma beta-carotene level, beta-carotene/retinol ratio and beta-carotene/total lipids ratio each showed significant correlations with serum HbA1c and fructosamine in all subjects studied. Similarly, the plasma alpha-tocopherol level and alpha-tocopherol/total lipids ratio were correlated with these indexes of glycemic control. These findings suggest certain mechanisms may exist to prevent lipid peroxidation and vascular complications in IDDM patients.

To investigate the antioxidant status of obese children, we analyzed beta-carotene and alpha-tocopherol levels in plasma and low density lipoprotein (LDL). We also analyzed the fatty acid composition of LDL as a substrate for oxidative stress. The plasma beta-carotene and alpha-tocopherol levels were relatively lower in obese girls than in normal controls. However, the plasma alpha-tocopherol/lipids ratio was significantly lower in obese girls than in normal controls. Both LDL beta-carotene and LDL alpha-tocopherol levels were significantly lower in obese girls than in normal controls, although no obvious differences were observed in plasma levels. In obese girls LDL contained more polyunsaturated fatty acid (PUFA) compared with normal controls. When the peroxidizability index (PI) was calculated to estimate the susceptibility of lipids to oxidative stress, obese girls had significantly higher PI values than normal controls. Both the LDL beta-carotene/PI ratio and the LDL alpha-tocopherol/PI ratio were significantly lower in obese girls than in normal controls. These results indicate the increased susceptibility of LDL to oxidative stress in obese girls which may promote atherosclerosis later in life.

Vesnarinone (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone), a quinolinone derivative, is an orally active inotropic agent used in Japan for the treatment of chronic heart failure. Recently, it has been reported that vesnarinone induces differentiation and apoptosis in certain types of leukaemia and solid tumour cells, and exhibits antitumour effect on several tumours xenografted in nude mice. In the present study, we examined the antitumour effect of vesnarinone in combination with radiation and conventional anticancer agents in nude mice xenografted with human gastric carcinoma, a poorly-differentiated adenocarcinoma, MKN-45 cell line which has a wild-type p53 gene. Vesnarinone treatment combined with radiation resulted in a higher antitumour activity compared with a single treatment with either vesnarinone or radiation alone. Further, vesnarinone treatment together with radiation and conventional anticancer agents including 5-FU and picibanil (an immunopotentiator) produced the highest antitumour effect compared with any other treatment. Additionally, the combination treatment induced marked differentiation and apoptosis of the tumour cells and an increase in the expression of p53 gene in the treated tumour cells. The results suggest that vesnarinone, in combination with radiation and the conventional antitumour agents, may be of clinical interest for treatment of certain types of gastric tumours.

The sites associated with dopamine neurons which produce mouse-killing aggression (muricide) were examined in the rat brain. Muricide appeared in 60-80% of rats after being fed a thiamine-deficient diet for 28 days. Microinjection of dopamine (500 ng/rat) into the olfactory bulb (OB) significantly suppressed muricide, whereas injection into other brain areas failed to do so. The incidence of muricide after dopamine injection was 40% at 5 min and 20% at 15-30 min. When 6-hydroxydopamine (8 micrograms/0.5 microliter), following pretreatment with desmethylimipramine (25 mg/kg i.p.), was injected twice into the ventral tegmental area (VTA) or the olfactory bulb (OB) in nonkiller rats during thiamine-deficient feeding, the occurrence of muricide gradually increased over time. The present results suggest that degeneration of dopamine neurons projecting from the VTA to the OB may be related to mouse-killing aggression in rats.

The relationship between learning and genetic factor on immobility in mice during a forced swimming test (FST) has been studied. The duration of immobility during the FST did not change significantly after the administration of either scopolamine (2.5 mg/kg, ip) or cyclohexamine (150 mg/kg, ip), although both drugs produced impairment in the learning task. This suggests that the increase in immobility observed during the second trial of the FST may not be related to learning which could occur during the first trial. Concerning the strain difference, first, the duration of immobility in C3H mice was shorter than that in ICR, ddY, C57BL and BALB mice. Second, after receiving shock stress in a box, ICR, ddY and C57BL mice, but not C3H mice showed a marked decrease in locomotor activity when placed in the box again without shock. Also during the FST, both C57BL and ddY mice, but not C3H mice showed prolongation of immobility and reduction in swimming after shock stress. The changes in locomotor activity, and immobility and swimming during the FST caused by shock stress in ddY mice recovered to normal levels after treatment with imipramine. From these results, it is suggested that the immobility shown during the FST, which may be independent of learning and dependent on some genetic factor, is a suitable model of depression in animals.

The in vivo induction of the differentiation of murine WEHI-3B D+ myelomonocytic leukemia cells was measured by flow cytometry, simultaneously staining leukemia cells for the marker exogenous beta-galactosidase and for differentiation by the antigen Mac-1 (CD11b/CD18). The WEHI-3B D+ leukemia cells were transfected with the E. coli lac-Z gene by electroporation and subclones that constitutively expressed high levels of the lac-Z gene product beta-galactosidase were established. Flow cytometric analyses of cells in the peritoneal cavities of mice bearing leukemia cells showed that cells continued to express beta-galactosidase for at least 14 days, and they were distinguishable from host-derived cells in vivo by their expression of the transfected gene. Simultaneous determination of the beta-galactosidase activity and Mac-1 content of cells in the peritoneal cavities of mice revealed that administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to the mice enhanced the expression of Mac-1 antigen by beta-galactosidase-positive cells. The results demonstrate that G-CSF may have clinical potential as a therapeutic differentiating agent, and that flow cytometric analysis provides a useful in vivo system to evaluate the therapeutic potential of agents capable of inducing terminal differentiation.

We investigated the effect of 5-aza-2'-deoxycytidine (ADC) (a specific inhibitor of DNA methylation) on differentiation, Lewisy (Ley) antigen expression, and DNA fragmentation (a biochemical marker of apoptosis) in human pancreatic cancer MIA PaCa-2 cells. ADC markedly inhibited the growth of MIA PaCa-2 cells. Flow cytometric analysis showed that ADC suppressed the cell population in the G1 phase, but enhanced it in the G2/M phase. On the other hand, several markers of cell differentiation including morphological and biochemical alterations were distinctly induced in cells treated with ADC. Morphologically, the cells were enlarged and thinner than the untreated control cells, and intracellular alkaline phosphatase (ALP) activity was markedly increased. Additionally, biochemical markers of apoptosis including DNA fragmentation and Ley antigen expression were induced in association with the appearance of morphological and biochemical markers of differentiation (ALP). These results suggest that the hypomethylation of DNA is involved in the molecular mechanism of growth inhibition, induction of apoptosis, and differentiation in human pancreatic cancer MIA PaCa-2 cells.

We examined the effect of vesnarinone, an oral cardiotonic, on the growth and differentiation of human myeloid leukemia cells. Vesnarinone alone markedly induced erythroid differentiation of HEL cells. All-trans-retinoic acid also induced erythroid differentiation of the cells, and the differentiation was greatly enhanced by combined treatment with vesnarinone and retinoic acid. HEL cells are highly resistant to some anticancer drugs, including vincristine, but treatment with vesnarinone greatly increased the sensitivity of HEL cells to vincristine. Enhancement of vincristine sensitivity by vesnarinone was not as significant for other leukemia cells. Expression of P-glycoprotein in HEL cells was effectively inhibited by vesnarinone, suggesting that the restoration of vincristine sensitivity is associated with decrease of P-glycoprotein expression in HEL cells. The plasma level of vesnarinone required to induce differentiation of leukemia cells is 30 micrograms/mL, which could be achieved with oral administration. These results suggest that vesnarinone should be useful in differentiation therapy for some types of myelogenous leukemia.

We recently developed a monoclonal antibody directed to carbohydrate antigen Le(y), BM-1/JIMRO, and found that expression of Le(y) antigen defined by BM-1/JIMRO was associated with the process of apoptosis, but not with cell proliferation or necrosis. In the present experiments, we examined with BM-1/JIMRO the effects of various differentiation inducers on the growth and expression of Le(y) antigen in human lung cancer A549 cells. We found that a specific inhibitor of methylation of DNA, 5-aza-2' deoxycytidine (ADC), could markedly induce expression of Le(y) antigen in association with induction of apoptosis and differentiation in the A549 cells. These results suggest that hypomethylation of DNA is involved in the molecular mechanisms of induction of Le(y) antigen, apoptosis and differentiation in the cells.