[Outcomes of Pharmacists' Involvement with Residents of Special Nursing Homes for the Elderly].

　The current study aimed to examine the outcomes of pharmacists' involvement with elderly people in special nursing homes. We analyzed 58 cases involving regular visits by community pharmacists to 41 residents. The residents' mean age was 87.8±6.9 years, and 68.3% were prescribed 6 or more types of medication. Antipsychotic and insomnia medication was taken by 24.4% and 31.8% of residents, respectively. Pharmaceutical consultation following medication use accounted for 60.3% of pharmacists' involvement with residents. The outcomes of these consultations included improvements in prescription content; the identification and prevention of adverse drug events; improvement in activities of daily living; and improvement in test results, sleep, and urination/bowel control. The results also suggested that pharmacists' intervention reduced drug costs. Information that facilitated involvement was most frequently acquired via conversations (67.2%) and conferences (24.1%) in the facilities. The most common information sources were care workers (72.4%), followed by nurses (37.9%), physicians (6.9%), and functional training instructors (6.9%). Information was also acquired from patients (3.4%) and their family members (5.2%). The findings indicated that regular visits by pharmacists to facilities for elderly people and conversations between residents, their family members, and physicians, nurses and various other professionals improved various pharmacotherapy outcomes.

A novel role for acinus and MCM2 as host-specific signaling enhancers of DNA-damage-induced apoptosis in association with viral protein gp70.

The interaction of viral proteins with host-cellular proteins elicits the activation of numerous cellular signal transduction pathways possibly leading to the viral pathogenesis. We previously demonstrated that infection with Friend leukemia virus (FLV) radiosensitizes murine hematopoietic cells via a p53-dependent apoptotic pathway in C3H hosts. Here, we show that the transduction of the env-gene (gp70) of Friend murine leukemia virus (F-MuLV) sensitized C3H-derived myeloid leukemia cells to DNA-damage (ionizing radiation as well as doxorubicin)-induced apoptosis through the activation of DNA-dependent protein kinase (DNA-PK) and P53. Knockdown of DNA-PK by siRNA inhibited the radiosensitization induced by gp70. In association with gp70 and DNA-PK, the acinus and MCM2 proteins were host-specifically overexpressed in C3H-derived cells. Taken together, these data suggested that gp70 enhances cellular DNA-damage-induced signaling in association with host-specific cellular proteins including acinus and MCM2 resulting in the activation of DNA-PK to phosphorylate P53. This in vitro study clearly indicates that the enhancement of DNA-damage-induced apoptosis by gp70 is not caused by the bone marrow environment of the host but is introduced by modified signaling in hematopoietic cells. The mechanisms involved in the ability of a viral protein to regulate cellular gene expression could provide invaluable insight into the manipulation of cellular pro-apoptotic signaling and the development of novel therapeutic strategies.

Comparison of murine gene expression profiles between spontaneous and radiation-induced myelogenous leukemias: stochastic and probabilistic expression variances in the former vs radiation-specific expression commonalities in the latter.

OBJECTIVE: To elucidate the common characteristics of murine radiation-induced myelogenous leukemias, global gene-chip expression profiles were compared with age-matched steady-state bone marrow tissue profiles and spontaneous myelogenous leukemia profiles. MATERIALS AND METHODS: Six each of C3H/He mice-derived radiation-induced and spontaneously developed myelogenous leukemias were analyzed. Bone marrow cells from five each of 2- and 21-month-old mice were used to subtract nonleukemic information in the analysis. mRNAs from individual mice were analyzed separately using 45,101 gene chips followed by computational biological analysis. RESULTS: First, principal component analysis (PCA) was performed to discriminate the gene expression profiles of individual mice with radiation-induced myelogenous leukemia from those of bone marrow cells from 2- or 21-month-old mice. Discriminant union genes for individual leukemias were then selected, which finally yielded 242 genes, among which six are radiation-related genes including Hus-1, Edf1a2, andVegf-c; 16 are apoptosis/cell-death-related genes, 13 are cell-cycle/cell-growth-related genes, and 50 are suppressor/promoter genes. PCA of these 242 genes consistently enabled the discrimination of the radiation-induced leukemias from the spontaneous leukemias. Second, the other components of the same PCA provided four different eigenvector clusters in an unsupervised manner representing four histopathological findings, with which the differential diagnosis in molecular taxonomy was significant as determined by analysis of variance of the global gene expression profiles. CONCLUSION: Discriminant union genes in radiation-induced myelogenous leukemias against spontaneous myelogenous leukemias and age-matched nonleukemic bone marrow profilings generated by unsupervised computational analysis essentially represent probabilistic biomarkers for radiation-induced myelogenous leukemias, which may contribute to developing a model for risk of secondary carcinogenesis in patients treated by whole-body irradiation.

Upregulation of c-myc gene accompanied by PU.1 deficiency in radiation-induced acute myeloid leukemia in mice.

OBJECTIVE: High-dose radiation exposure induces acute myeloid leukemia (AML) in C3H mice, most of which have a frequent hemizygous deletion around the D2Mit15 marker on chromosome 2. This region includes PU.1, a critical candidate gene for initiation of leukemogenesis. To identify novel cooperative genes with PU.1, relevant to radiation-induced leukemogenesis, we analyzed the copy number alterations of tumor-related gene loci by array CGH, and their expressions in primary and transplanted AMLs. MATERIALS AND METHODS: For the induction of AMLs, C3H/He Nrs mice were exposed to 3 Gy of x-rays or gamma-rays. The genomic alterations of 35 primary AMLs and 34 transplanted AMLs obtained from the recipient mice transplanted the primary AMLs were analyzed by array CGH. According to the genomic alterations and mutations of the 235th arginine of PU.1 allele, we classified the radiogenic AMLs into three types such as Chr2(del) PU.1(del/R235-) AML, Chr2(del) PU.1(del/R235+) AML and Chr2(intact) PU.1(R235+/R235+) AML, to compare the expression levels of 8 tumor-related genes quantitatively by real-time polymerase chain reaction and cell-surface antigen expression. Results. In addition to well-known loss of PU.1 with hemizygous deletion of chromosome 2, novel genomic alterations such as partial gain of chromosome 6 were recurrently detected in AMLs. In this study, we found similarity between cell-surface antigen expressions of bone marrows and those of spleens in AML mice and significantly higher expressions of c-myc and PU.1 expression, especially in the PU.1-deficient (Chr2(del) PU.1(del/R235-)) AML and Chr2(del) PU.1(del/R235+) compared to Chr2(intact) PU.1(R235+/R235+) AMLs. CONCLUSION: The new finding on upregulation of c-myc and PU.1 in both and hemizygous PU.1-deficient AMLs and different genomic alterations detected by array CGH suggests that the molecular mechanism for development of radiation-induced AML should be different among three types of AML.

Intestinal tumorigenicity of mice carrying hemizygous Pax6, Pax6(Sey-4H).

The genotype-phenotype relationship in mice was examined experimentally using one of the small eye mutants, Pax6(Sey-4H), which deletes the chromosome 2 middle region, hemizygously. The genotyping indicated that the deleted region starts at a site 102.60 Mb from the centromere and has a length of 6.51 Mb, in which 12 known and 27 novel genes are located. Expecting the development of myeloid leukemia, gamma-irradiation was performed to female mutants at the age of 10 weeks. The mutants did not develop myeloid leukemia during the observation period of 18 months. Instead, they developed tumors in the alimentary tract spontaneously (56.0%). The tumor latency was shortened by the radiation exposure, but the tumor incidence of the gamma-irradiated group (62.5%) was as high as that of spontaneously developing tumors. Intraductal proliferation of the epithelium of the Wirsung duct was observed in the gamma-irradiated mutants (18.8%). Considering the results of the Pax6(Sey-4H) mutant together with those of another small eye mutant, Pax6(Sey-3H), the anomaly and the tumorigenicity of the intestinal tract were closely related to the hemizygosity of the 3.2 Mb segment of chromosome 2, where both mutants show a common deletion.

Direct detection of p53 -/- thymocyte appearing at an early stage of radiation-induced thymic lymphomagenesis in p53 +/- heterozygous B10 mice.

PURPOSE: The appearance of tumor suppressor protein 53 (p53) -/- thymocytes at an early stage of radiation-induced lymphomagenesis was investigated in the p53 heterozygous (+/-) B10 mice following a single dose of irradiation, since most thymic lymphomas manifested the loss of the wild-type p53 allele and the loss of heterozygosity was thought to be an early event critical for radiation-induced thymic lymphomagenesis in p53 +/- mice. MATERIALS AND METHODS: The mice were exposed to a single dose (6 Gy) of irradiation to induce thymic lymphomas and, at various times after irradiation, treated with an extremely high dose (30 Gy) of whole-body irradiation to enrich p53 -/- thymocytes and, 24 h later, the remaining thymocytes were assayed for cell surface markers and p53 genotype. RESULTS: In a significant fraction of the p53 +/- mice 5 weeks after 6 Gy irradiation, there was a relative increase in the number of cluster of differentiation (CD) 4+CD8+ thymocyte subpopulation among thymocytes remaining after 30 Gy irradiation. The CD4+CD8+ double-positive (DP) thymocytes were shown to contain p53-/- cells, and the number of p53 -/- thymocytes was more than 10(5) in those individuals. CONCLUSIONS: The results clearly indicated that an extremely high dose (30 Gy) of whole-body irradiation enabled us to directly detect p53 -/- thymocytes in an abundant p53 +/- thymocyte population and that proliferative p53 -/- thymocytes develop in a CD4+CD8+ DP thymocyte subpopulation within a few weeks after a single dose (6 Gy) of irradiation.

p53 (TRP53) deficiency-mediated antiapoptosis escape after 5 Gy X irradiation still induces stem cell leukemia in C3H/He mice: comparison between whole-body assay and bone marrow transplantation (BMT) assay.

Mice exposed to a lethal dose of radiation were repopulated with heterozygous p53(+/-) (TRP53(+/-)) bone marrow cells and then exposed to doses of 1, 3 and 5 Gy 1 month later. This resulted in the transplanted bone marrow-specific diseases other than competitively induced nonhematopoietic neoplasms. Interestingly, the present study showed a high frequency of stem cell leukemia, i.e., leukemias characterized by a lack of differentiation due also to p53 deficiency, even after 5 Gy irradiation. The frequencies of stem cell leukemias (and those of total hematopoietic malignancies) were 16% (24%) at 1 Gy and 45% (75%) at 3 Gy. Furthermore, markedly high incidences of stem cell leukemias were observed at 5 Gy in p53(+/-) mice, i.e., 87% (100%) in the transplantation assay and 60% (83.3%) in the whole-body assay, whereas a conventional whole-body assay induced only 14% in wild-type mice. The high incidence of stem cell leukemias observed in this study using heterozygous p53-deficient mice agrees with results of a previous study of homozygous p53-deficient mice and is consistent with the high frequency of loss of heterozygosity in the p53 wild-type allele observed in leukemias. This suggests that the target cells for radiation-induced stem cell leukemias may be p53-deficient hematopoietic stem cells.

Caloric restriction prevents radiation-induced myeloid leukemia in C3H/HeMs mice and inversely increases incidence of tumor-free death: implications in changes in number of hemopoietic progenitor cells.

OBJECTIVES: Previously, we found a clear decrease in the incidence of radiation-induced myeloid leukemia in C3H/HeMs mouse caused by caloric restriction (CalR). In this report, CalR before and after irradiation was examined to determine whether they exert different effects on the prevention of radiation-induced myeloid leukemogenesis and the consequent extension of life span by CalR. METHODS: The C3H/HeMS strain, which is prone to radiation-induced myeloid leukemia, was used. Groups subjected to different CalR timings, pre- and postirradiation, were compared with groups not subjected to CalR during their lifetime for the incidences of neoplasms, specifically that of myeloid leukemia, and the incidence of tumor-free death. A single dose of 3Gy X-ray was administered to mice at 10 weeks old. Results of colonization assay before and after CalR were compared with the incidence of leukemogenesis among the groups. RESULTS: Irrespective of the CalR timing in terms of irradiation, there was a significant difference in the prevention of myeloid leukemogenesis, and a consequent difference in longevity (731 approximately 805 days for CalR groups vs. 697 days for the group without CalR; Log rank, P<0.03). During CalR, the number of hemopoietic progenitor cells (HPCs), potential leukemogenic targets, significantly decreased (0.4 x 10(4) vs. 4.2 x 10(4) of granulomacrophage colony forming units per spleen; 1.3 x 10(4) vs. 7.6 x 10(4) of the splenic colony forming units per spleen), but this decreased number of HPCs returned to that of the non-CalR control group, when the CalR group was returned to nonrestricted diet (returned to 1.5 x 10(4) granulomacrophage colony-forming units per spleen; returned to 2.8 x 10(4) splenic colony-forming units per spleen). Although preirradiation CalR followed by a conventional non-CalR diet negates the potential preventive effect, prevention conferred by pre-and postirradiation CalR suggests different underlying mechanisms; preirradiation CalR prevents the initiation of direct genotoxic leukemogenesis, while postirradiation CalR the indirect, epigenetic, leukemogenesis. CONCLUSION: The incidences of tumor-free death significantly increased in all the groups undergoing CalR except for the group subjected to preirradiation CalR, which contributed to the longevity of the groups undergoing CalR.

Mutations of the PU.1 Ets domain are specifically associated with murine radiation-induced, but not human therapy-related, acute myeloid leukaemia.

Murine radiation-induced acute myeloid leukaemia (AML) is characterized by loss of one copy of chromosome 2. Previously, we positioned the critical haematopoietic-specific transcription factor PU.1 within a minimally deleted region. We now report a high frequency (>65%) of missense mutation at codon 235 in the DNA-binding Ets domain of PU.1 in murine AML. Earlier studies, outside the context of malignancy, determined that conversion of arginine 235 (R235) to any other amino-acid residue leads to ablation of DNA-binding function and loss of expression of downstream targets. We show that mutation of R235 does not lead to protein loss, and occurs specifically in those AMLs showing loss of one copy of PU.1 (P=0.001, Fisher's exact test). PU.1 mutations were not found in the coding region, UTRs or promoter of human therapy-related AMLs. Potentially regulatory elements upstream of PU.1 were located but no mutations found. In conclusion, we have identified the cause of murine radiation-induced AML and have shown that loss of one copy of PU.1, as a consequence of flanking radiation-sensitive fragile domains on chromosome 2, and subsequent R235 conversion are highly specific to this mouse model. Such a mechanism does not operate, or is extremely rare, in human AML.

DNA-dependent protein kinase enhances DNA damage-induced apoptosis in association with Friend gp70.

Friend leukemia virus (FLV) infection strongly enhances gamma-irradiation-induced apoptosis of hematopoietic cells of C3H hosts leading to a lethal anemia. Experiments using p53 knockout mice with the C3H background have clarified that the apoptosis is p53-dependent and would not be associated with changes of cell populations caused by the infection with FLV. In bone marrow cells of FLV + total body irradiation (TBI)-treated C3H mice, the p53 protein was prominently activated to overexpress p21 and bax suggesting that apoptosis-enhancing mechanisms lay upstream of p53 protein in the signaling pathway. Neither of DNA-dependent protein kinase (DNA-PK)-deficient SCID mice nor ataxia telangiectasia mutated (ATM) gene knockout mice with the C3H background exhibited a remarkable enhancement of apoptosis or p53 activation on FLV + TBI-treatment indicating that DNA-PK and ATM were both essential. ATM appeared necessary for introducing DNA damage-induced apoptosis, while DNA-PK enhanced p53-dependent apoptosis under FLV-infection. Surprisingly, viral envelope protein, gp70, was co-precipitated with DNA-PK but not with ATM in FLV + TBI-treated C3H mice. These results indicated that FLV-infection enhances DNA damage-induced apoptosis via p53 activation and that DNA-PK, in association with gp70, might play critical roles in modulating the signaling pathway.

Spontaneous and radiation-induced leukemogenesis of the mouse small eye mutant, Pax6(Sey3H).

Allelic loss on the chromosome 2 is associated with radiation-induced murine acute myeloid leukemia. However, the gene, which contributes mainly to the leukemogenesis has not yet been identified. Expecting any predisposition to acute myeloid leukemia, we performed a radiation leukemogenensis experiment with Pax6(Sey3H), one of the small eye mutants carrying a congenital hemizygosity of the chromosome 2 middle region. A deletion mapping of Pax6(Sey3H) with 50 STS markers indicated that the deleted segment extended between the 106.00 and 111.47 Mb site from the centromere with a length of 5.47 Mb. In the deleted segment, 6 known and 17 novel genes were located. Pax6(Sey3H) mutants that crossed back into C3H/He did not develop myeloid leukemia spontaneously, but they did when exposed to gamma-rays. The final incidence of myeloid leukemia in mutants (25.8%) was as high as that in normal sibs (21.4%). Survival curves of leukemia-bearing mutants shifted toward the left (p = 0.043 by the Log rank test). F1 hybrids of Pax6(Sey3H) with JF1 were less susceptible to radiation than Pax6(Sey3H) onto C3H/He in regard to survival (p = 0.003 and p < 0.00001 for mutants and normal sibs, respectively, by a test of the difference between two proportions). Congenital deletion of the 5.47 Mb segment at the middle region on chromosome 2 alone did not trigger myeloid stem cells to expand clonally in vivo; however, the deletion shortcut the latency of radiation-induced myeloid leukemia.

Retrotransposition of limited deletion type of intracisternal A-particle elements in the myeloid leukemia Clls of C3H/He mice.

The murine genome has about 1,000 copies of DNA elements for the intracisternal A-particle (IAP) that resembles a retrovirus. We previously reported that the genomic DNA of the cells from radiation-induced acute myeloid leukemia (AML) lines derived from C3H/He inbred mice was frequently rearranged by the integration of the IAP element. In this study, 8 IAP elements from the characteristic integration sites in 6 cell lines of radiation-induced AML from different mice were characterized and compared in structure with 114 IAP elements isolated from the normal C3H/He genome. One of the 8 elements was a full-length type I IAP, and 7 were of type-I Delta 1 with a common deletion site. Although the type I Delta 1 form is a minor population accounting for about 6% of total genomic IAP elements, it is predominantly retrotransposed in the AML cells from different C3H/He mice. This indicates that limited populations of the IAP elements contribute to the unique retrotransposition in AML cells.

Evaluation of nonthreshold leukemogenic response to methyl nitrosourea in p53-deficient C3H/He mice.

The classic controversy of whether genotoxic chemicals induce cancers with or without a certain low-dose limit, i.e., the threshold, is revisited because of a number of current publications available addressing the plausibility of "practical" thresholds even for genotoxic carcinogens, the mechanism of which may be hypothesized to be due, in part, to a repair system composed of ordinarily available various defense mechanisms under the steady-state DNA damage. The question of whether an absolute nonthreshold or a relative nonthreshold, i.e., a "practical" threshold specifically in the low-dose level, is present may not be answered even with the use of a prohibitively large number of wild-type mice. Could the excessive incidence of tumorigenesis in p53-deficient mice contribute to our understanding of the threshold vs nonthreshold issue in genotoxic carcinogenesis? This is considered because an exaggeration of tumorigenesis in p53-deficient mice is hypothesized to reduce or eliminate the range of threshold due to the p53-deficiency-mediated reduction of DNA repair and apoptosis. The present study of chemical leukemogenesis in p53-deficient mice by transplantation assay was designed to answer this question. Briefly, 218 C3H/He mice were lethally irradiated and repopulated with bone marrow cells from wild-type, heterozygous p53-deficient, and homozygous p53-deficient C3H/He mice. This was followed by treatment with a single and graded dose of methyl nitrosourea at 6.6, 14.8, 33.3, 50.0, and 75.0 mg/kg body wt, with the vehicle-treated control groups treated with zero dose for each genotype. Whereas mice repopulated with p53-deficient bone marrow cells showed a marked reduction of the threshold for leukemogenicity, mice repopulated with wild-type bone marrow cells did not exhibit leukemia at a dose of 33.3 mg/kg body wt and showed a curve with a high probability for the linear regression model with a positive dose intercept, predicting a threshold by the likelihood ratio test. Thus, the failure of wild-type mice to show an increase in incidence of leukemogenesis at low doses of genotoxic carcinogens may be due not to a statistical rarity, but to various p53-related pharmacophysiological functions, possibly including DNA repair and apoptosis that may account for a threshold.

Stem-cell leukemia: p53 deficiency mediated suppression of leukemic differentiation in C3H/He myeloid leukemia.

C3H/He mice produce myeloid leukemias after whole body irradiation of 1-3Gy as compared with non-irradiated controls that produce fewer than 1% of leukemia [Radiatiton Research 127 (1991) 146]. Thus, p53-deficient C57BL/6 strain, a malignant lymphoma prone, was crossed back into C3H/He strain. Lethally irradiated wild-type mice to which p53-deficient bone marrow cells were transplanted (transplantation assay) showed dramatic change in the propensity of leukemia of myeloid lineages, the cells lacking CD3, Thy1.2, sIgM, B220, Mac-1, Gr-1, but being positive for c-Kit and CD44. Furthermore, transplanted mice subjected to 3Gy irradiation gave rise to a faster development of leukemia and a higher frequency of double-lineage leukemias than the non-irradiated control.

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.

Cytogenetic analysis of radiation-induced leukemia in Trp53-deficient C3H/He mice.

C3H/He mice develop acute myeloid leukemia (AML) after whole-body irradiation, but the strain becomes highly susceptible to stem cell leukemia (SCL) when a null mutation is introduced into the Trp53 gene. To examine the etiology of SCL and the influence of chromosomal instability on leukemogenesis, 12 SCLs and two AMLs arising from Trp53-deficient C3H/He mice were investigated cytogenetically. Each SCL demonstrated cell-to-cell variation in the number and structural integrity of their chromosomes, indicating chromosomal instability. Typical deletion of chromosome 2 was observed in the two AML cases, while most SCL cells did not display this aberration. Deletions and rearrangements of chromosome 11 were noticeable in SCLs from Trp53 heterozygotes but not in AMLs. Analysis of loss of heterozygosity revealed that aberrations involving chromosome 11 in SCLs resulted in loss of the wild-type Trp53 allele. These results suggest that loss of Trp53 function triggers the tumorigenic process leading toward SCL through the induction of chromosomal instability, and that SCL and AML are distinct varieties of leukemia.