SMC protein complexes and the maintenance of chromosome integrity.

Structural maintenance of chromosomes (SMC) family proteins have attracted much attention for their unique protein structure and critical roles in mitotic chromosome organization. Elegant genetic and biochemical studies in yeast and Xenopus identified two different SMC heterodimers in two conserved multiprotein complexes termed 'condensin' and 'cohesin'. These complexes are required for mitotic chromosome condensation and sister chromatid cohesion, respectively, both of which are prerequisite to accurate segregation of chromosomes. Although structurally similar, the SMC proteins in condensin and cohesin appear to have distinct functions, whose specificity and cell cycle regulation are critically determined by their interactions with unique sets of associated proteins. Recent studies of subcellular localization of SMC proteins and SMC-containing complexes, identification of their interactions with other cellular factors, and discovery of new SMC family members have uncovered unexpected roles for SMC proteins and SMC-containing complexes in different aspects of genome functions and chromosome organization beyond mitosis, all of which are critical for the maintenance of chromosome integrity.

Mutations/deletions of the WT1 gene, loss of heterozygosity on chromosome arms 11p and 11q, chromosome ploidy and histology in Wilms' tumors in Japan.

Incidence rates of Wilms' tumor (WT) markedly differ in East Asian and Caucasian children. In the present study, we examined WT1 deletions/mutations and loss of heterozygosity (LOH) on 11p and 11q in a large number of WTs and compared our findings with those from 4 series of Caucasian WTs. Incidence rates of the subtle WT1 mutation in 3 of the 5 series of sporadic and unilateral WTs including ours were 4.3-6.2% and similar. However, gross homozygous WT1 deletion was more frequent in our series than in some others. In addition, our series tended to show a higher incidence of LOH limited to 11p13 and a lower incidence of LOH including 11p15 than the Caucasian one. These findings indicate some genetic differences in WT between the 2 regions. One of the 4 Caucasian series reported a correlation of germinal WT1 mutation with the predominantly stromal histology. The present study not only confirms the correlation of germinal WT1 deletion/mutation with predominant stromal histology but also establishes a correlation with somatic WT1 deletion/mutations with predominant stromal histology. While WTs with WT1 abnormalities usually showed pseudodiploidy and predominant stromal histology, those without WT1 abnormalities showed various chromosome numbers and histologic subtypes.

Cancer-prone syndrome of mosaic variegated aneuploidy and total premature chromatid separation: report of five infants.

Five infants (two girls and three boys) from four families all had severe pre- and postnatal growth retardation, profound developmental delay, microcephaly, hypoplasia of the brain with Dandy-Walker complex or other posterior fossa malformations, and developed uncontrollable clonic seizures. Four infants developed Wilms tumors, and one showed cystic lesions in bilateral kidneys. All five infants showed variegated mosaic aneuploidy in cultured lymphocytes. In two infants whose chromosomes were prepared by us, 48.5%-83.2% lymphocytes showed total premature chromatid separation (PCS). Their parents had 3.5%-41.7% of their lymphocytes in total PCS. The remaining three infants and their parents, whose chromosomes were prepared at outside laboratories, tended to show lower frequencies of total PCS. Another five infants reported with the disorder were reviewed together with the five infants we described. Together, their clinical and cytogenetic manifestations were similar enough to suggest a syndrome. Seven of the 10 infants developed proven or probable Wilms tumors. The age at diagnosis of the tumors was younger than usual at 2-16 months. The tumors were bilateral in four infants and unilateral in three infants, and cystic changes were present in six infants. Two infants developed botryoid rhabdomyosarcoma. The carriers of the syndrome are thus liable to tumorigenesis. The possible role of mitotic checkpoint defects, proven in two infants with the syndrome (Matsuura et al. [2000: Am J Hum Genet 69:483-486]), was discussed in connection with tumor development and progression.

A potential role for human cohesin in mitotic spindle aster assembly.

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to form in vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.

Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A.

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

The structural maintenance of chromosomes (SMC) family of proteins in mammals.

Structural maintenance of chromosomes (SMC) family proteins play critical roles in chromosome structural changes. SMC proteins are known to be involved in two major chromosome structural organization events required for mitotic segregation of chromosomes: mitotic chromosome condensation and sister chromatid cohesion. In eukaryotes, two separate sets of SMC heterodimers form the cores of two distinct multiprotein complexes termed 'condensin' and 'cohesin', each specialized for condensation or cohesion, respectively. It is clear that both condensin and cohesin are conserved in mammals, including humans. The mammalian complexes demonstrate dynamic changes in intracellular distribution in a cell cycle-dependent manner. At any point in the cell cycle, the intracellular localization of the majority of mammalian cohesin and condensin appears to be complementary. Cohesin is associated with chromatin in interphase, while condensin is largely cytoplasmic. Similarly, in mitosis, cohesin is mostly excluded from chromosomes while condensin is distinctly bound to them. Cell cycle-dependent targeting of the two complexes appears to play a major role in regulating their cell cycle-specific activities, and how this redistribution is controlled is an area of active research. Finally, there is evidence that SMC proteins may be involved in DNA recombination and repair. This review focuses on what we have learned about SMC family proteins in humans and other mammalian species in comparison to those in lower eukaryotes. The authors present their own views with regard to some of the major outstanding questions surrounding the nature and functions of the SMC family of proteins.

A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation.

Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E was determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.

Expression and imprinting status of human PEG8/IGF2AS, a paternally expressed antisense transcript from the IGF2 locus, in Wilms' tumors.

A large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes.

Competitive polymerase chain reaction for the determination of N-myc amplification in neuroblastoma: report of clinical cases.

If an unfavorable prognosis is suspected in neuroblastoma, decision on a treatment protocol should be based on the N-myc copy number (12). We already demonstrated that the newly developed competitive polymerase chain reaction (competitive PCR) is a promising method for the determination of the N-myc copy number (6), and have started to use this competitive PCR procedure in neuroblastoma patients, together with fine-needle biopsy in selected cases. Seven children were studied. In one infant of 5 months of age whose tumor was diagnosed before undergoing mass screening for neuroblastoma, the competitive PCR procedure was performed with a fine-needle biopsy, and after obtaining a negative report on N-myc amplification within 48 hours, a regular protocol of treatment could be started without delay. We report that competitive PCR is a rapid and accurate method for the determination of the N-myc copy number, requiring only a small amount of material, and anticipate that competitive PCR will become the procedure of choice for the determination of N-myc copy number in neuroblastoma.

A new technique applicable to pediatric laparoscopic surgery: abdominal wall 'area lifting' with subcutaneous wiring.

BACKGROUND: Recently, the authors developed a unique method of laparoscopic surgery without pneumoperitoneum: "area lifting of the abdominal wall with subcutaneous wiring." METHODS: In this gasless procedure, the anterior abdominal wall is pulled upward by a pair of wires placed subcutaneously and held by thick sutures for "hanger lifting." Simultaneous lifting of a pair of subcutaneous wires across the abdomen, produces a wide, roof-shaped intraabdominal space sufficient for laparoscopic surgical procedures. The practical aspects of this gasless technique, as well as the authors' limited experience with this method in 24 children, ranging from 8 days to 15 years of age is presented. These children have had various pathologies including splenomegaly, rectal prolapse, ovarian cyst, gall stone, adrenal neuroblastoma, and abdominal wall abscess. CONCLUSIONS: Gasless laparoscopic surgery with double subcutaneous wiring is safe for children including neonates and those with respiratory compromise because all operative procedures are performed under normal abdominal pressure. Because of the highly elastic abdominal wall musculature inherent in children, this selective area lifting of abdominal wall creates a relatively larger peritoneal volume than in adults.

Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics.

The structural maintenance of chromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and Xenopus. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the Xenopus chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription.

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Extensive genetic heterogeneity in the neuroblastoma cell line NB(TU)1.

A neuroblastoma cell line displaying genetically unique features was established from a stage III case of a 20-month-old girl. Southern blotting by the probe pTNB6, which contains exon 1 of the N-myc gene, showed that the primary tumor had in total 4 aberrant bands beside the normal amplified band. The established cell line NB(TU)1 had an aberrant N-myc band (9.0 kb) in addition to the normal band (2.9 kb). Cytogenetic analysis revealed that NB(TU)1 has a composite karyotype composed of at least 7 related karyotypes, which are pseudo-diploid and contain complex chromosomal abnormalities, including translocations, deletions and homogeneously staining regions (HSRs). Such extensive abnormalities were considered to be prominent among known neuroblastoma cell lines, and it was suggested that NB(TU)1 had acquired a certain type of genetic instability. Analysis of N-myc bands in 11 clones of NB(TU)1 showed that the intensity ratio of the normal-sized band (2.9 kb) and the aberrant one (9.0 kb) markedly varied among clones. Moreover, 3 clones showed an additional band with the size of 3.7 kb, which was detectable neither in the parent NB(TU)1 nor in the primary tumor. Thus, NB(TU)1 was shown to be composed of heterogeneous cell components. To further detect such ongoing chromosomal instability, we examined micronuclei formation. NB(TU)1 yielded a larger number of micronuclei than 5 other neuroblastoma cell lines. We conclude that NB(TU)1 has acquired genetic instability detectable by both Southern blotting and cytogenetic analysis.

Infrequent mutations of the TP53 gene and no amplification of the MDM2 gene in hepatoblastomas.

We have investigated the mutation of the TP53 gene in hepatoblastomas (HBLs) by using polymerase chain reaction-single strand conformation polymorphism and direct sequencing in 38 HBL tumor samples and in two HBL cell lines. We detected the TP53 gene mutation in an anaplastic hepatoblastoma cell line, but no aberration of the TP53 gene (exons 5-9) was found in tumor samples and in the other HBL cell line. The mutation of the cell line was a missense mutation from GAC (asparagine) to CAC (histidine) at codon 281, which was different from the G-to-T transversion of codon 249 that is frequently found in adult hepatocellular carcinomas (HCCs). In addition, we performed Southern blot analysis of the MDM2 gene, but we did not find MDM2 gene amplification in 19 cases tested. Our results suggest that, in contrast to the findings in HCCs in adults, TP53 gene aberrations are not involved in the development or progression of HBLs in children.

Drosophila TFIIA-S is up-regulated and required during Ras-mediated photoreceptor determination.

Photoreceptor induction in the developing Drosophila eye is triggered by the activation of the Ras pathway. Subsequently, the Ras-mediated activation of site-specific transcription factors leads to the expression of putative "effector" genes. The coactivator function of the basal transcription factor TFIIA has been shown previously to enhance the trans-activation potential of site-specific transcription factors in vitro. Here, we show that the expression of the small subunit of TFIIA (dTFIIA-S) is specifically up-regulated in a transient manner during Ras-mediated photoreceptor induction. Furthermore, although null mutations in dTFIIA-S are cell lethal, a hypomorphic dTFIIA-S allele demonstrates an increased requirement for this factor during photoreceptor development. In addition, the cone cell to R7 photoreceptor transformation caused by ectopic activation of the Ras pathway during eye development is suppressed by the removal of one functional copy of the dTFIIA-S locus revealing the sensitivity of this process to reductions in dTFIIA-S activity. These results are the first in vivo evidence for the coactivator function in transcriptional enhancement proposed for TFIIA.

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus.

Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low-stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules.

Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression.

The hemagglutinin-esterase (HE) protein of mouse hepatitis virus (MHV) is an optional envelope protein present in only some MHV isolates. Its expression is regulated by the copy number of a UCUAA pentanucleotide sequence present in the leader sequence of the viral genomic RNA. The functional significance of this viral protein so far is not clear. In this report, we compared the neuropathogenicity of two MHV isolates, JHM(2) and JHM(3), which express different amounts of HE protein. Intracerebral inoculation of these two viruses into C57BL/6 mice showed that JHM(2), which expresses an abundant amount of HE protein, was more neurovirulent than JHM(3), which expresses very little HE. Histopathology showed that early in infection, JHM(2) infected primarily neurons, while JHM(3) infected mainly glial cells. JHM(3) eventually infected neurons and caused a delayed death relative to JHM(2)-infected mice, suggesting that the progression of JHM(3) infection in the central nervous system was slower than JHM(2). In vitro infection of JHM(3) in primary mixed glial cell cultures of astrocyte-enriched cultures yielded higher virus titers than JHM(2), mimicking the preferential growth of JHM(3) in glial cells in vivo. These findings suggest that the reduced neuropathogenicity of JHM(3) may correlate with its preferential growth in glial cells. Sequence analysis showed that the S genes of these two viruses are identical, thus ruling out the S gene as the cause of the difference in neuropathogenicity between these two viruses. We conclude that the HE protein contributes to viral neuropathogenicity by influencing either the rate of virus spread, viral cell tropism or both.

[Hereditary renal tumors: Wilms' tumor--congenital anomalies' syndrome].

The genetics and associated abnormalities of Wilms' tumor are reviewed. Wilms' tumor is associated with several congenital syndromes such as WAGR (Wilms' tumor, aniridia, genitourinary malformation, mental retardation) syndrome, Denys-Drash syndrome, Beckwith-Wiedemann syndrome, etc. However, the association with such syndromes is relatively infrequent and accounts for less than 5% of all clinical patients with Wilms' tumor. WAGR syndrome and Denys-Drash syndrome are associated with loss of WT1 gene located in the chromosome 11p13, and BW syndrome is considered to be due to duplication of the paternal 11p15 allele (WT2). The association of Wilms' tumor with primary brain tumors in a daughter and a mother is also described.