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Objective: On the basis of the hypothesis that copy number mutations of the genes encoding myelin compact proteins are responsible for myelin disorders in humans, we have explored the possibility of copy number mutations in patients with Charcot-Marie-Tooth disease (CMT) whose responsible genes remain undefined., Methods: A family with 6 affected members in 3 consecutive generations, presenting with motor and sensory demyelinating polyneuropathy, was investigated. Characteristic clinical features in this pedigree include Adie pupils and substantial intrafamilial variability in the age at onset, electrophysiological findings, and clinical severity. Nucleotide sequence analyses of PMP22, MPZ, or GJB1 and gene dosage study of PMP22 did not reveal causative mutations. Hence, we applied a custom-designed array for comparative genomic hybridization (CGH) analysis to conduct a comprehensive screening of copy number mutations involving any of the known causative genes for CMT other than PMP22., Results: The array CGH analyses revealed increased gene dosage involving the whole MPZ, and the flanking genes of SDHC and C1orf192. The gene dosage is estimated to be 5 copies. This mutation showed complete cosegregation with the disease phenotype in this pedigree., Interpretation: The increased gene dosage of MPZ and increased expression level of MPZ mRNA emphasize the important role of the dosage of the MPZ protein in the functional integrity of peripheral nerve myelin in humans, and provide a new insight into the pathogenic mechanisms underlying CMT., (Copyright © 2011 American Neurological Association.)

To clarify genome-wide DNA methylation profiles during hepatocarcinogenesis, bacterial artificial chromosome (BAC) array-based methylated CpG island amplification was performed on 126 tissue samples. The average numbers of BAC clones showing DNA hypo- or hypermethylation increased from noncancerous liver tissue obtained from patients with hepatocellular carcinomas (HCCs) (N) to HCCs. N appeared to be at the precancerous stage, showing DNA methylation alterations that were correlated with the future development of HCC. Using Wilcoxon test, 25 BAC clones, whose DNA methylation status was inherited by HCCs from N and were able to discriminate 15 N samples from 10 samples of normal liver tissue obtained from patients without HCCs (C) with 100% sensitivity and specificity, were identified. The criteria using the 25 BAC clones were able to discriminate 24 additional N samples from 26 C samples in the validation set with 95.8% sensitivity and 96.2% specificity. Using Wilcoxon test, 41 BAC clones, whose DNA methylation status was able to discriminate patients who survived more than 4 years after hepatectomy from patients who suffered recurrence within 6 months and died within a year after hepatectomy, were identified. The DNA methylation status of the 41 BAC clones was correlated with the cancer-free and overall survival rates of patients with HCC. Multivariate analysis revealed that satisfying the criteria using the 41 BAC clones was an independent predictor of overall outcome. Genome-wide alterations of DNA methylation may participate in hepatocarcinogenesis from the precancerous stage, and DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication., (Copyright (c) 2009 UICC.)

Gastric cancer is the second common malignant neoplasia in Japan, and its poorly differentiated form is a deadly disease. To identify novel candidate oncogenes contributing to its genesis, we examined copy-number alterations in 50 primary poorly differentiated gastric cancers using an array-based comparative genomic hybridization (array-CGH). Many genetic changes were identified, including a novel amplification of the 13q22 locus. Several genes are located in this locus, and selective knockdown of one for the Krüppel-like factor 12 (KLF12) induced significant growth-arrest in the HGC27 gastric cancer cell line. Microarray analysis also demonstrated that genes associated with cell proliferation were mostly changed by KLF12 knockdown. To explore the oncogenic function of KLF12, we introduced a full length of human KLF12 cDNA into NIH3T3 and AZ-521 cell lines and found that overexpression significantly enhanced their invasive potential. In clinical samples, KLF12 mRNA in cancer tissue was increased in 11 of 28 cases (39%) when compared with normal gastric epithelium. Clinicopathological analysis further demonstrated a significant correlation between KLF12mRNA levels and tumor size (p = 0.038). These data suggest that the KLF12 gene plays an important role in poorly differentiated gastric cancer progression and is a potential target of therapeutic measures.

Objective: Recently, genomic multiplications of alpha-synuclein gene (SNCA) have been reported to cause hereditary early-onset parkinsonism. The objective of this study was to assess the frequency of SNCA multiplications among autosomal dominant hereditary Parkinson's disease (ADPD)., Methods: We screened 113 ADPD probands and 200 sporadic PD cases by quantitative polymerase chain reaction and confirmed SNCA multiplications by fluorescence in situ hybridization (FISH) and comparative genomic hybridization array., Results: Two families (two patients from Family A and one from Family B) with SNCA duplication were identified among ADPD patients. Even though they had the same SNCA duplication, one patient had dementia. Because there was exactly the same difference between the regions originated from each patient, the finding suggests that the phenotype of SNCA multiplication may be also influenced by the range of duplication region. We also detected asymptomatic carriers in the families of both patients. Interestingly, the penetrance ratio was 33.3% (2/6) in one kindred, indicating that the ratio was very much lower than expected., Interpretation: These two newly identified Japanese patients with SNCA duplication and the five previously identified American and European families with SNCA triplication or duplication mutations indicate that the incidence of SNCA multiplication may be more frequent than previously estimated.

Comparative genomic hybridization (CGH) was used to screen for changes in the number of DNA sequence copies in 30 primary colorectal cancers and 16 liver metastases, to identify regions that contain genes important for the development and progression of colorectal cancer. In primary colorectal cancer, we found frequent gains at 7p21 (36.7%), 7q31-36 (30%), 8q23-24 (43.0%), 12p (30%), 14q24-32 (33.3%), 16p (40.0%), 20p (33.3%), 20q (63.3%) and 21q (36.3%), while loss was often noted at 18q12-23 (36.7%). In metastatic tumors, there were significantly more gains and losses of DNA sequences than in primary tumors, with gains at 8q23-24 (found in 62.5% of recurrences vs. 43.0% of primary tumors), 15q21-26 (37.5% vs. 20.0%), 19p (43.8% vs. 20.0%) and 20q (81.3% vs. 63.3%) and losses at 18q12-23 (50.0% vs. 36.7%). The pattern of genetic changes seen in metastatic tumors, with frequent gains at 8q23-24 and 20q and loss at 18q12-23, suggests the progression of colorectal cancer. We investigated a clinical follow-up study for all patients examined by CGH and directed our attention to the genetic changes consisting of gains at 8q and 20q. The incidence of liver metastases was higher in patients with primary colorectal cancer with these genetic changes. Gains at 8q and 20q might be useful to identify patients at high risk for developing liver metastases., (Copyright 2001 Wiley-Liss, Inc.)

Comparative genomic hybridization studies have revealed frequent amplification of the 14q12-q13 region in esophageal squamous cell carcinoma (ESC) cell lines. To identify genes targeted for amplification, we first defined the minimal common region of amplification using fluorescence in situ hybridization in affected ESC cell lines. The amplicon covered about 6 Mb, between markers D14S1034 and L18528. Then we screened 32 ESC cell lines to discern amplifications and expression levels of 26 expressed sequence tags (ESTs) that had been localized to the amplified region. Five known genes (BAZ1A, SRP54, NFKBIA, MBIP, and HNF3A) and two uncharacterized ESTs (GenBank Accession numbers AA991861 and AA167732) within the amplicon showed amplification and consequent overexpression. Two of these transcripts were amplified in three of the primary ESCs we examined. Our findings suggest that these seven genes are candidate targets of the amplification mechanism and therefore may be associated, together or separately, with development and progression of ESC., (Copyright 2001 Wiley-Liss, Inc.)

We performed molecular analysis of a germline interstitial deletion of chromosome 4 [del(4)(q21.22q23)], which had been observed in a male infant manifesting early-onset hepatoblastoma (HBL). The chromosomal anomaly in this child was associated with a unique congenital syndrome including HBL, atrial septal defect, ventricular septal defect, patent ductus arteriosus, mental retardation, and seizures. However, the patient did not exhibit a megalencephaly typical of 4q21-22 deletions. His HBL was associated with an increasing serum alpha-fetoprotein level and rapid growth. To define the chromosomal deletion at the molecular level in this child, we analyzed his lymphoblasts with fluorescence in situ hybridization, using as probes a panel of BAC/PAC genomic clones containing STS markers covering the 4q12-27 region. The analysis revealed that the affected chromosome had an 8-cM deletion within 4q21-q22, flanked by markers D4S2964 and D4S2966. This microdeletion overlaps with the commonly deleted region at 4q21-q22 that was recently defined in adult hepatocellular carcinomas., (Copyright 2001 Wiley-Liss, Inc.)

Recent molecular studies have shown a relatively high rate of loss of heterozygosity (LOH) in neuroblastoma (NB) as well as other types of tumors in human chromosome band 1p36. To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in NB cell lines with PCR according to a high-density sequence tagged site (STS)-content map spanning 1p35-36. Among 25 NB cell lines examined, only one cell line, NB-1, showed no signal with 27 STSs in a 480 kb region in 1p36.2. The sequence analysis has revealed that the defective region included seven known genes (E4, KIF1B, SCYA5, PGD, Cortistatin, DFF45, and PEX14), nine expressed sequence tags (ESTs), and two microsatellite markers. These genes are related to apoptosis, an ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule, and components of a common translocation machinery. The region between the DFF45 and KIF1B genes was defined as homozygous deletion by Southern blotting. The search in LOH regions with high-density STSs may be useful for the isolation and identification of tumor suppressor genes in other tumors as well as NBs., (Copyright 2001 Wiley-Liss, Inc.)

Background: We have identified for the first time a homozygously deleted region within the smallest region of overlap at 1p36.2-3 in two neuroblastoma cell lines., Procedure: The 800-kb PAC contig covering the entire homozygously deleted region was made and sequenced. To date, approximately 70% of sequencing has been accomplished, and the estimated length of the deleted region was 500 kb., Results: Currently, we have found six genes within the region, which include three known genes as well as three other genes that have been reported during processing of our present project for the last 3(1/2) years. We report here the results of expression and mutation analyses of those genes., Conclusions: Full sequencing for the region of homozygous deletion as well as further analyses of the genes mapped within the region may reveal whether or not there is a neuroblastoma suppressor gene as proposed by the Knudson's two-hit hypothesis., (Copyright 2000 Wiley-Liss, Inc.)

Classical cytogenetic approaches have revealed many of the chromosomal aberrations that may occur in gastric cancers (GC), although few alterations of specific genes have been identified so far. Genes that affect progression of this disease need to be identified if clinicians are to achieve optimal management of patients with GC. As the first step toward the cloning of gene(s) that may be involved in gastric carcinogenesis, we examined 25 GC cell lines for aberrations in DNA copy number to detect chromosomal gains and losses, as well as gene amplifications, by comparative genomic hybridization (CGH). Our CGH study revealed high-level amplifications in chromosomal regions that had been well defined in GC but also in sites that had not, including 3p24, 5p15, 11p11.2-14, 13q34, 15q26, Xp24, and Xq26-28. The minimal common region at 11p13, within the 11p11.2-14 amplicon, harbors the CD44 gene. Northern and Western blot analyses showed that an alternatively spliced form of CD44, with variant exons 8-10 (CD44E), was overexpressed in all cell lines bearing the 11p13 amplicon. However, cell adhesion activity was no greater in these lines than in cell lines without amplifications at the CD44 locus, suggesting that the major property of upregulated CD44 in these cases might be to transduce signals critically associated with growth and proliferation of the tumor cells., (Copyright 2000 Wiley-Liss, Inc.)

Our analysis of chromosomal aberrations in primary gastric cancers using comparative genomic hybridization has revealed novel, high and frequent copy number increases in the long arm of chromosome 20, indicating that this region contains novel amplified genes involved in gastric cancer progression. AIB1, a member of the steroid receptor co-activator-1 family, has been cloned on 20q12 as a candidate target gene for this amplification in human breast cancers. In this study, we examined the numbers of AIB1 copies as well as their expression and relation to clinico-pathological features in 72 primary gastric cancers. AIB1 amplification was observed in 7% and over-expression in 40% of the specimens. AIB1 amplification always coincided with its over-expression, but several cases showed AIB1 over-expression without amplification, suggesting that expression of AIB1 is regulated not only by gene amplification but also by other mechanisms, such as transcriptional activation, in human gastric cancer. Gastric cancers with AIB1 amplification showed extensive lymph node metastases, liver metastases and poorer prognosis compared to those without amplification. Our results suggest that amplification and over-expression of AIB1 are likely to increase the number of malignant phenotypes of gastric cancers and that it can be expected to be useful as a marker of poor prognosis., (Copyright 2000 Wiley-Liss, Inc.)

Spectral karyotyping (SKY) is a new molecular cytogenetic technique that allows simultaneous visualization of each chromosome in a different color. We have used SKY for comprehensive analysis of 20 myelodysplastic syndromes (MDSs) (13 primary MDSs, 3 therapy-related MDSs, and 4 acute leukemias developed from MDS, including 1 cell line established from a secondary leukemia), previously analyzed by G-banding. To locate the chromosomal breakpoints, DAPI-counterstained band images from all metaphases were transformed to G-band-like patterns. By using SKY, it was possible to identify the origin and organization of all clonal marker chromosomes (mar), as well as the origin of all abnormalities defined as additional material of unknown origin (add) or homogeneously staining regions (hsr) by G-banding. In total, SKY identified the chromosomal basis of 38 mar, add, and hsr, corrected 8 abnormalities misidentified by G-banding, and revealed 6 cryptic translocations in 5 cases. Total or partial chromosomal loss (mainly of -5/5q- and -7/7q-) is the most frequent cytogenetic abnormality in MDS. In 3 of 11 cases with -5/5q- and in 4 of 8 with -7/7q-, lost material was detected by SKY in unbalanced translocations. A total of 60 chromosomal losses were identified by G-banding in 16 cases with multiple chromosome abnormalities involving at least 3 chromosomes. For 26 of these losses (43%), SKY analysis suggested that the losses were not complete, but had been translocated to a variety of partner chromosomes. Moreover, SKY analysis revealed that a ring chromosome in a case of acute leukemia developed from MDS contained three to six segments that originated from chromosome 21 material. Fluorescence in situ hybridization showed the amplification of the AML1 gene on regions derived from chromosome 21, providing the first evidence of amplification involving this gene in MDS. Genes Chromosomes Cancer 26:336-345, 1999., (Copyright 1999 Wiley-Liss, Inc.)

We investigated copy number aberrations in 29 primary tumors and 12 cell lines of esophageal squamous cell carcinoma (ESC) using comparative genomic hybridization. In the primary tumors, the most common sites of copy number gains were 3q26.3-27 (45%), 8q24 (41%), 5p15 (38%), Xq27-28 (38%), 14q32 (31%), 11q13 (28%), and 20q13.3 (28%). High-level gains (HLGs) indicative of gene amplifications were identified at 11q13 in two cases, and in one case each at 2q33-34, 3q25-29, 5p15.1-15.2, 7q21-22, 11p11.2, 12p11.2-12, and 13q34. Recurrent losses were observed only at 9p13(17.2%). In the 12 ESC cell lines, the most common sites of HLGs were 5p15.1-15.3 (four cases), 11q13 (four cases), 8q24.1-24.2 (three cases), 20q13.2-13.3 (three cases), 3q26.3 (two cases), and 7p15-22 (two cases). Less frequent HLGs (one case each) were observed at 2p16-22, 3q25, 7p12-14, 7q21-22, 9q34, 10q21, 11p11.2, 14q13-14, 14q31-32, 15q22-26, and 17p11.2. Chromosomes and chromosome arms that showed frequent losses in the cultured lines were 18q (58%), 4 (50%), 9p (50%), and 3p (42%). These findings provide evidence for a number of previously unknown genomic aberrations in ESC, suggesting target regions for positional cloning of genes relevant to carcinogenesis in the esophagus. In particular, we identified a significant amplification of the DPI gene (TFDPI), a transcription factor that forms heterodimers with E2FI, in the single primary tumor that exhibited HLG at 13q34.

By means of comparative genomic hybridization (CGH), we screened 58 primary gastric cancers for changes in copy number of DNA sequences. We detected frequent losses on Ip32-33 (21%), 3p21-23 (22%), 5q14-22 (36%), 6q16 (26%), 9p21-24 (22%), 16q (21%), 17p13 (48%), 18q11-21(33%), and 19(40%). Gains were most often noted at I p36 (22%), 8p22-23 (24%), 8q23-24 (29%), 11q12-13 (24%), 16p(21%), 20p (38%), 20q (45%), Xp21-22(38%), and Xq21-23 (43%), with high-level amplifications at 6p21(2%),7q31(10%), 8p22-23(5%), 8q23-24 (7%), 11q13(4%), 12p12-13(4%), 17q21(2%), 19q12-13(2%), and 20q13(2%). High-level amplification at 8p22-23 has never been reported in any other cancer type and its frequency was as high as that reported for the MYC, MET, and KRAS genes. We narrowed down the smallest common amplicon to 8p23.1 by reverse-painting FISH to prophase chromosomes. Southern blot analysis using one EST marker (D38736) clearly demonstrated that amplification of this exon-like sequence had occurred in all three tumors in which amplifications at 8p22-23 had been detected by CGH. Our data provide evidence for several, previously undescribed, genomic aberrations that are characteristic of gastric cancers.

Partial-tandem duplication (PTD) of an internal portion of MLL occurs in some cases of acute myelogenous leukemia (AML) with trisomy 11 or a normal karyotype. This type of MLL rearrangement may be transcribed into an mRNA species that is capable of encoding a partially duplicated protein associated with leukemogenesis. However, although several kinds of oncogenes, especially MYC, are often amplified on double-minute chromosomes (dmins) in hematological malignancies, no amplification of MLL has been reported in AML. Here, we report the first documented case of a patient with AML whose leukemic cells exhibited amplification of MLL on dmins. Furthermore, in this patient, MLL was rearranged in a PTD manner, with in-frame fusion of exons 2 and 6.

Smith-Magenis syndrome (SMS) is caused by a microdeletion of 17p11.2 and comprises developmental and growth delay, facial abnormalities, unusual behavior and sleep problems. This phenotype may be due to haploinsufficiency of several contiguous genes. The human brain finger protein gene (ZNF179), a member of the RING finger protein family, has been isolated and mapped to 17p11.2. FISH analyses of metaphase or interphase chromosomes of 6 patients with SMS show that ZNF179 was deleted in one of the 2 homologs (17p11.2), indicating a possible association of the defect of this gene with the pathogenesis of SMS. Furthermore, using a prophase FISH ordering system, we sublocalized ZNF179 proximally to LLGL which lies on the critical region for SMS.

Background: Several studies have recently demonstrated that apoptosis of cancer cells is triggered by diverse adjuvant cancer therapies and the induction of apoptosis correlates with the sensitivity of the primary tumor to such therapies., Methods: We investigated the factors modulating adjuvant cancer therapies by examining p53 gene mutations and chromosome 17p allelic losses in 15 rectal cancers treated by a preoperative combined therapy consisting of radiation, intraluminal hyperthermia and 5-fluorouracil suppositories., Results: The point mutations were detected in 7 of 15 (46.7%) tumors by single-stranded conformational polymorphism and direct sequencing. Allelic losses at chromosome 17p were also detected in 7 of 15 (46.7%) tumors by dinucleotide-repeat polymorphisms. There was no correlation between p53 gene abnormalities and the preoperative tumoricidal effect of the therapy., Conclusions: We conclude that p53 gene abnormalities do not directly increase resistance to the combined adjuvant therapy.

We undertook a cytogenetic analysis of 29 human brain tumors using double-target fluorescence in situ hybridization (FISH) and focusing on chromosome arm 1p. One or more tumor suppressor genes in this arm have been suggested to be important in a variety of neuroectodermal tumors. The series included 9 oligodendrogliomas, 4 mixed gliomas, 10 astrocytomas, 4 glioblastomas, and 2 central neurocytomas. We hybridized pericentromeric (1q12) and subtelomeric (1p36) DNA probes to cell nuclei prepared from paraffin-embedded tissues and observed a strikingly high incidence of deletion of at least part of 1p in oligodendrogliomas (100%) and mixed gliomas (75%). The results of the FISH analyses were confirmed by demonstration of loss of heterozygosity for a microsatellite polymorphism in 10 of the 29 tumors. As well as supporting the feasibility of FISH for detecting allelic deletions in chromosomes from paraffin-embedded tumor samples, the alteration of 1p reported here will contribute to an understanding of the molecular genetic events in oligodendroglial tumor development.

We have constructed a physical map of chromosome band 17p13, using 29 markers that had been localized to 17p13 by means of fluorescence in situ hybridization (FISH) and analysis by pulsed-field gel electrophoresis (PFGE). The map spans nearly 8 Mb of genomic DNA, and the estimated average distance between each marker is roughly 290 kb. The p13 band of chromosome 17 is thought to contain a putative tumor suppressor gene in addition and distal to TP53. Deletion mapping in a large number of breast carcinomas indicated that the tumor suppressor gene lies between the loci defined by cC117-708 (D17S878) and p144D6 (D17S34), which are an estimated 7 Mb apart. Our results should contribute to construction of a contig map of chromosome band 17p13 with cosmid and/or YAC (yeast artificial chromosome) clones, and to isolation of the putative tumor suppressor gene.

A detailed cytogenetic map was constructed around the chromosomal breakpoint of t(8;13) observed in a patient with multiple exostoses. The order of seven loci defined by cosmid clones mapped to 8q23 was determined by means of two-color fluorescence in situ hybridization (FISH) on elongated prophase chromosomes, and localizations of these markers relative to the breakpoint were examined. The results indicated that loci defined by cC18-553 and cC18-1512 flank the breakpoint. By pulsed-field gel electrophoresis of DNA digested with BssHII and Southern hybridization with cC18-1512, DNA from the patient showed a band which was not observed in DNA isolated from either parent. As the normal size of this BssHII fragment is 600 kb, the chromosomal breakpoint probably lies less than 600 kb away from cC18-1512.

We have investigated the mRNA expression of 2 human protein tyrosine phosphatases with sequence homology to cytoskeletal proteins, PTPH1 and PTPMEG. Northern-blot analysis of PTPH1 using poly (A)+ RNA from normal human colon tissue showed a low-abundance message of 4.3 kb. Reverse-transcriptase/polymerase-chain reaction (RT-PCR) was therefore used to detect it in a wide variety of cell lines including 9 colorectal, 5 gastric, 5 hepatic and 6 hematopoietic tumor cells. PTPH1 mRNA was not detected only in Colo 320 cells over-expressing c-myc mRNA, among the colorectal cancer cell lines examined. When Colo 320 cells were incubated with 5 mM sodium butyrate for 5 days, PTPH1 mRNA became detectable, concomitant with the marked decrease in the expression level of c-myc mRNA. Moreover, the chromosomal localization of PTPH1 gene was investigated by fluorescence in situ hybridization. Interestingly, PTPH1 gene was mapped to 9q31 where the gene for Gorlin syndrome, a putative tumor suppressor gene, exists.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by development of bilateral acoustic neurinomas and increased incidence of meningiomas. Frequent losses of I allele of chromosome 22 in neurinomas and meningiomas has indicated that the gene responsible for NF2 functions as a tumor suppressor. Although the NF2 gene has been mapped within a 13 cM region between D22S1 and D22S28 by linkage analysis, its location with respect to D22S15 is uncertain. We previously reported an NF2 patient with a constitutional balanced translocation t(4;22)(q12;q12.2); the NF2 gene is probably disrupted at the breakpoint. To define the location of this breakpoint on chromosome 22, we performed fluorescence in situ hybridization (FISH) with DNA markers in the NF2 region and determined the physical order of 5 loci: D22S1-NF2-LIF-D22S15-D22S32.

The neural crest gives rise to a variety of tissues, including peripheral neurons, Schwann cells, melanocytes and ectomesenchymal cells, which include the smooth-muscle cells of large arteries. Cell lines derived from neuroblastoma (a neural-crest tumor) exhibit at least 2 distinct morphological cell types, a neuroblastic phenotype (N-type) and an epithelial-like phenotype (S-type) with characteristics of substrate-adhesiveness. We have analyzed 17 human neuroblastoma cell lines using a panel of monoclonal antibodies (MAbs) against cytoskeletal proteins. Three neuroblastoma cell lines (KP-N-SI, KP-N-YN and SMS-KCN) bound an alpha-smooth-muscle actin antibody. In addition, one of these lines (KP-N-SI) bound anti-desmin MAbs as determined by indirect immunofluorescence. A total of 8 cloned cell lines were obtained from the above parent cell lines. These were composed of either N- or S-type cells and were confirmed to have the same neuroblastoma origin as each parent cell line by chromosomal analysis. Alpha-smooth-muscle actin and desmin were demonstrated in the S-type cloned cells by indirect immunofluorescence, as well as by 2-dimensional Western blot analysis. These results were confirmed by Northern blot analysis using a specific probe (pSH alpha SMA-3' UT) to human alpha-smooth-muscle actin mRNA. These ascertain the presence of alpha-smooth-muscle actin and desmin in neuroblastoma cell lines. These data show that, in addition to giving rise to cells with neural, Schwann-cell and melanocyte markers, neuroblastoma can also give rise to the cells expressing smooth-muscle cell markers.

Drug resistance is one of the major impediments to the treatment of advanced neuroblastoma. Two neuroblastoma cell lines established from the same patient before (KP-N-AY) and after (KP-N-AYR) chemotherapy are described. Both cell lines were established from bone-marrow metastases of a 2 1/2-year-old patient with stage IV neuroblastoma. Chromosomal analysis, catecholamine assessment and the surface membrane phenotype of these cell lines confirmed that the tumors were of neuroblastoma origin. Compared with the KP-N-AY cell line, the KP-N-AYR line had decreased N-myc amplification but increased N-myc expression. An in vitro sensitivity test using a clonogenic assay showed the KP-N-AYR cell line to be 3.0-fold resistant to adriamycin and 2.7-fold resistant to cis-platinum as compared with the KP-N-AY cell line. The expression of the multi-drug-resistance gene (MDR1) was not observed in either cell line by the ribonuclease protection assay. The KP-N-AY cell line revealed only faint MDR1 RNA by the polymerase chain reaction, whereas the KP-N-AYR cell line had no expression of the MDR1 gene. The level of glutathione-S-transferase-pi was significantly higher in the KP-N-AYR cell line than in the KP-N-AY cell line. These findings suggest that the development of clinical drug resistance may be associated with the enhanced glutathione-S-transferase-pi activity but not with MDR1 gene expression.

A parent rhabdomyosarcoma cell line designated SCMC-RM2 was established from bone-marrow tumor cells taken from an 11-year-old girl with an embryonal rhabdomyosarcoma. Subsequently a cloned SCMC-RM2-1 cell line was isolated from a parent line. These cell lines grew as adherent monolayers in liquid culture with a doubling time of 50 and 52 hr, respectively. In addition, colonies were established in soft agar, which grew in a dose-dependent fashion with a cloning efficiency of 0.7 and 0.8%, respectively. Chromosomal analysis showed these cell lines had neither double minutes nor homogeneously staining regions. Chromosome number ranged from 61 to 93, translocation; t(9;13)(p22;q14) was identified, and no alteration of chromosome 2 was observed. Surface membrane antigen profile of parent and cloned lines by using a panel of 24 monoclonal antibodies (MAbs) excluded the possibility of these being neuroblastoma cell lines. In addition, MAbs to the cytoplasmic protein desmin, myoglobin, muscle actin (alpha and gamma) and alpha-sarcomeric actin reacted with these cell lines, SCMC-RM2 and SCMC-RM2-1 being thus identified as rhabdomyosarcoma. Southern blot analyses revealed 8- and 7-fold amplification of the N-myc gene in SCMC-RM2 and SCMC-RM2-1 as compared with the promyelocytic cell line HL60. Over-expression of the N-myc mRNA was noted over control cell lines.

We established the subline RT-BMV-C6 from the parent human neuroblastoma cell line RT-BM by a process that required repeated subculture of cells, which were prone to disaggregation. RT-BMV-C6 and the parent cloned line, RT-BM-1, had an identical marker chromosome, confirming that both lines were derived from a common progenitor. In the analysis of surface antigen expression, RT-BMV-C6 did not react with UJ-127-11, Leu7 or KP-NAC2 MAbs to which RT-BM-1 showed positive binding. The levels of both N-myc amplification and expression in RT-BMV-C6 were twice as high as the level obtained in RT-BM-1. Colony-forming efficiency in soft agar was 2.0 +/- 0.8% for RT-BMV-C6 and 3 times greater than that for RT-BM-1 (0.6 +/- 0.1%). When 100 x 10(6) cells of RT-BM-1 and RT-BMV-C6 were inoculated into nude mice, tumor incidence was significantly higher for RT-BMV-C6 (6/6; 100%) than for RT-BM-1 (0/6; 0%). Our data show that N-myc is closely related to tumorigenicity in NB. When RT-BM-1 and RT-BMV-C6 were co-cultured with a new synthetic retinoid, polyprenoic acid (E5166), and dibutyryl cyclic AMP, RT-BM-1 was induced to neuronal differentiation, defined by the formation of neuronal processes and expression of neurofilaments, whereas RT-BMV-C6 was not. However, when exposed to E5166, N-myc expression of RT-BMV-C6 was more strongly reduced than that of RT-BM-1, and colony formation of RT-BMV-C6 was significantly inhibited as compared to RT-BM-1. These findings suggest that the reduction of N-myc expression might closely correlate with growth inhibition accompanying neuronal differentiation of neuroblastoma cells.