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Biomedical subjects

J A Huberman

Publications and source records attributed to J A Huberman.

At least 19 recordsLinked to original sources
PubMed2001

MCM2 is an independent predictor of survival in patients with non-small-cell lung cancer.

PURPOSE: Minichromosome maintenance protein 2 (MCM2) is a component of the prereplicative complex. It is essential for eukaryotic DNA replication and is only expressed in proliferating cells. The prognostic utility of MCM2 compared with Ki-67, another marker of proliferating cells, on survival of patients with non-small-cell lung cancer (NSCLC) was studied. PATIENTS AND METHODS: We examined the immunohistochemical expression of MCM2 and Ki-67 in primary pathologic tumor specimens from 221 NSCLC patients. For each marker, the fraction of tumor cells with positive staining was assessed as a percentage and categorized into four groups: 0% to 24%, 25% to 49%, 50% to 74%, and > or = 75%. MCM2 and Ki-67 immunoreactivities were compared with each other, and associations with pathologic and clinical parameters predictive of survival were analyzed with the chi(2) test. Cox regression models were used to assess associations between MCM2 and Ki-67 and survival while controlling for confounders. RESULTS: Independent variables significantly associated with survival were tumor stage, performance status, and staining category. Patients with less than 25% MCM2 immunoreactivity had a longer median survival time than patients with > or = 25% MCM2 immunoreactivity (46 v 31 months; P =.039) and a lower relative risk (RR) of death (RR, 0.55, 95% confidence interval, 0.34 to 0.88). There was no significant association between survival and Ki-67 expression. CONCLUSION: Immunostaining of tumor cells for MCM2 is an independent prognostic parameter of survival for patients with NSCLC. Interpretable results can be obtained on more than 96% of paraffin-embedded specimens, and approximately 35% will be in the favorable subgroup, with less than 25% positively stained tumor cells. Whether MCM2 is predictive of response to therapy needs to be studied.

Adenocarcinoma↗
PubMed2001

Regulation of replication timing in fission yeast.

Here we report the first characterization of replication timing and its regulation in the fission yeast Schizosaccharomyces pombe. We used three different synchronization methods: centrifugal elutriation, cdc10 temperature-shift and release, and starvation for deoxyribonucleoside triphosphates (dNTPs) by treatment with hydroxyurea (HU) followed by removal of HU, to study the times when specific autonomously replicating sequence elements (ARS elements; potential replication origins) replicate during S phase. We found that individual ARS elements replicate at characteristic times, some early and some late, independently of synchronization method. In wild-type cells treated with HU, early ARS elements replicated but late ones did not. However, in HU-treated mutant cells lacking the Rad3 (similar to human ATR and ATM) or Cds1 (similar to human CHK2) checkpoint kinase, both early and late ARS elements were able to replicate. Thus under conditions of dNTP starvation the Rad3 and Cds1 kinases are needed to suppress the replication of normally late-replicating regions.

Adenosine Triphosphatases↗
PubMed2001

MCM2--a promising marker for premalignant lesions of the lung: a cohort study.

BACKGROUND: Because cells progressing to cancer must proliferate, marker proteins specific to proliferating cells may permit detection of premalignant lesions. Here we compared the sensitivities of a classic proliferation marker, Ki-67, with a new proliferation marker, MCM2, in 41 bronchial biopsy specimens representing normal mucosa, metaplasia, dysplasia, and carcinoma in situ. METHODS: Parallel sections were stained with antibodies against MCM2 and Ki-67, and the frequencies of staining were independently measured by two investigators. Differences were evaluated statistically using the two-sided correlated samples t-test and Wilcoxon rank sum test. RESULTS: For each of the 41 specimens, the average frequency of staining by anti-MCM2 (39%) was significantly (p < 0.001) greater than by anti-Ki-67 (16%). In metaplastic lesions anti-MCM2 frequently detected cells near the epithelial surface, while anti-Ki-67 did not. CONCLUSIONS: We conclude that MCM2 is detectable in 2-3 times more proliferating premalignant lung cells than is Ki-67. The promise of MCM2 as a sensitive marker for premalignant lung cells is enhanced by the fact that it is present in cells at the surface of metaplastic lung lesions, which are more likely to be exfoliated into sputum. Future studies will determine if use of anti-MCM2 makes possible sufficiently early detection to significantly enhance lung cancer survival rates.

Biomarkers, Tumor↗
PubMed2001

Multiple redundant sequence elements within the fission yeast ura4 replication origin enhancer.

BACKGROUND: Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized. RESULTS: Systematic deletion of consecutive segments of approximately 50, approximately 100 or approximately 150 bp within the enhancer and its adjacent core origin (ars3002) revealed that several of the approximately 50-bp stretches within the enhancer contribute to its function in partially redundant fashion. Other stretches within the enhancer are inhibitory. Some of the stretches within the enhancer proved to be redundant with sequences within core ars3002. Consequently the collection of sequences important for core origin function was found to depend on whether the core origin is assayed in the presence or absence of the enhancer. Some of the important sequences in the core origin and enhancer co-localize with short runs of adenines or thymines, which may serve as binding sites for the fission yeast Origin Recognition Complex (ORC). Others co-localize with matches to consensus sequences commonly found in fission yeast replication origins. CONCLUSIONS: The enhancer within the ura4 origin cluster in fission yeast contains multiple sequence motifs. Many of these stimulate origin function in partially redundant fashion. Some of them resemble motifs also found in core origins. The next step is to identify the proteins that bind to these stimulatory sequences.

Journal Article↗
PubMed2001

Roles for internal and flanking sequences in regulating the activity of mating-type-silencer-associated replication origins in Saccharomyces cerevisiae.

ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette. Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own. We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins. We could not detect consistent differences in sequence requirements for the two types of origins. Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity. Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase. In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p.

Cell Cycle Proteins↗
PubMed2000

Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci.

According to the current paradigm, replication foci are discrete sites in the interphase nucleus where assemblies of DNA replication enzymes simultaneously elongate the replication forks of 10-100 adjacent replicons (each approximately 100 kbp). Here we review new results and provide alternative interpretations for old results to show that the current paradigm is in need of further development. In particular, many replicons are larger than previously thought - so large that their complete replication takes much longer (several hours) than the measured average time to complete replication at individual foci (45-60 min). In addition to this large heterogeneity in replicon size, it is now apparent that there is also a corresponding heterogeneity in the size and intensity of individual replication foci. An important property of all replication foci is that they are stable structures that persist, with constant dimensions, during all cell cycle stages including mitosis, and therefore likely represent a fundamental unit of chromatin organization. With this in mind, we present a modified model of replication foci in which many of the foci are composed of clusters of small replicons as previously proposed, but the size and number of replicons per focus is extremely heterogeneous, and a significant proportion of foci are composed of single large replicons. We further speculate that very large replicons may extend over two or more individual foci and that this organization may be important in regulating the replication of such large replicons as the cell proceeds through S-phase.

Animals↗
PubMed1999

Induction by adozelesin and hydroxyurea of origin recognition complex-dependent DNA damage and DNA replication checkpoints in Saccharomyces cerevisiae.

DNA damaging agents induce a conserved intra-S-phase checkpoint that inhibits DNA replication in eukaryotic cells. To better understand this checkpoint and its role in determining the efficacy of antitumor drugs that damage DNA, we examined the effects of adozelesin, a DNA-alkylating antitumor agent that has a profound inhibitory effect on initiation of DNA replication in mammals, on the replication of Saccharomyces cerevisiae chromosomes. Adozelesin inhibited initiation of S. cerevisiae DNA replication by inducing an intra-S-phase DNA damage checkpoint. This inhibitory effect was abrogated in orc2-1 cells containing a temperature-sensitive mutation in a component of the origin recognition complex (ORC) that also causes a defect in initiation. The orc2-1 mutation also caused a defect in a checkpoint that regulates the activation of origins in late S phase in cells treated with hydroxyurea. Defects in both initiation and checkpoint regulation in the orc2-1 strain were suppressed by deletion of a gene encoding a putative acetyltransferase, SAS2. Adozelesin also induced a cellular response that requires a function of ORC in G(1). A similar G(1)-specific response in mammals may contribute to the cytotoxic and antitumor properties of this and other DNA-damaging drugs.

Antineoplastic Agents, Alkylating↗
PubMed1999

Influence of a replication enhancer on the hierarchy of origin efficiencies within a cluster of DNA replication origins.

DNA replication origins in animal cells sometimes occur in clusters. Often one of the multiple origins within these clusters fires more frequently than the others. The reason for this hierarchy remains unknown. Similar origin clusters occur in the fission yeast, Schizosaccharomyces pombe. One such cluster is located near the ura4 gene on chromosome III and contains three origins: ars3002, ars3003, and ars3004. In their natural chromosomal context (ars3003 is about 2.5 kb upstream of ars3002 and ars3004 is adjacent to ars3002 on the downstream side) their initiation frequencies display a striking hierarchy: ars3002 >> ars3003 >> ars3004. Here, we describe experiments that reveal a 400 bp replication enhancer within ars3004, adjacent to ars3002. The enhancer is essential for ars3004 origin function in a plasmid, but even with the enhancer ars3004 is an inefficient origin. The enhancer is not essential for ars3002 plasmid origin activity, but dramatically stimulates this activity, converting ars3002 from an inefficient plasmid origin to a very efficient one. It also stimulates the plasmid origin activity of ars3001 and ars3003 at all tested positions and orientations on both sides of each autonomously replicating sequence (ARS) element. If ars3002 is redefined to include the enhancer, then the relative activities of the three ARS elements as single origins within separate plasmids or as origins when all three ARS elements are present in a single plasmid is the same as the chromosomal hierarchy. Thus, this replication enhancer defines the relative activities of the three origins in the ura4 origin region. Similar enhancers may affect relative activities in the origin clusters of animal cells.

Cell Count↗
PubMed1999

Genetic methods for characterizing the cis-acting components of yeast DNA replication origins.

Small circular plasmids containing replication origins and, in some cases, centromeres, can replicate autonomously in the nuclei of all tested yeast species. Because this autonomous replication is dependent on the replication origin within the plasmid, measurements of the efficiency of autonomous replication (by the methods summarized here) permit evaluation of the effects of mutations on origin function. Although alternative methods are available for genetic characterization of replication origins in other organisms, the simplicity of the autonomous replication assay in yeasts has permitted development of the deepest understanding to date of eukaryotic replication origin structure. This information has come primarily from studies with Saccharomyces cerevisiae. However, there are many other yeast species, each with its own variety of replication origins. Use of the methods summarized here to characterize origins in other yeast species is likely to provide additional insights into eukaryotic replication origin structure.

Cell Division↗
PubMed1999

DNA damage and replication checkpoints in the fission yeast, Schizosaccharomyces pombe.

Eukaryotic organisms have developed an array of mechanisms for minimizing the consequences of damage to their DNA molecules and the consequences of interference with their DNA replication. Among these mechanisms are the DNA damage and replication checkpoints, which inhibit passage from one cell cycle stage to the next when DNA is damaged or replication is incomplete. Studies of these checkpoints in the fission yeast, Schizosaccharomyces pombe, complement studies in other organisms and provide valuable insight into the nature of the proteins responsible for these checkpoints and how such proteins may function.

DNA Damage↗
PubMed1998

Ribosomal DNA replication in the fission yeast, Schizosaccharomyces pombe.

We have employed genetic and two-dimensional (2D) gel electrophoretic methods to identify replication initiation, pausing, and termination sites in the tandem ribosomal DNA (rDNA) repeats of the fission yeast, Schizosaccharomyces pombe. An autonomously replicating sequence (ARS) element, ars3001, maps to a 2.3-kb restriction fragment spanning the junction between the nontranscribed spacer (NTS) and the external transcribed spacer upstream of the ribosomal RNA genes, and 2D gel analysis shows that replication initiates in the NTS portion of the same fragment. A pause region at the 3' end of the rRNA genes inhibits forks from entering these genes counter to the direction of transcription. Thus, most forks move through the genes in the same direction as transcription. In these respects, fission yeast rDNA replication resembles that in the budding yeast, Saccharomyces cerevisiae, and in multicellular eukaryotic organisms. A feature which, so far, has been detected only in fission yeast is the pausing of replication forks in a broad region near the 5.8S rRNA gene.

DNA Replication↗
PubMed1998

Multiple orientation-dependent, synergistically interacting, similar domains in the ribosomal DNA replication origin of the fission yeast, Schizosaccharomyces pombe.

Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp). Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs. In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small ( approximately 570 bp) and responsible for replication of ribosomal DNA. Like previously studied fission yeast origins, ars3001 contains multiple important regions. The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other. These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins. It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast.

Base Sequence↗
PubMed1998

Isolation, characterization, and molecular cloning of a protein (Abp2) that binds to a Schizosaccharomyces pombe origin of replication (ars3002).

The autonomously replicating sequence (ARS) element ars3002 is associated with the most active replication origin within a cluster of three closely spaced origins on chromosome III of Schizosaccharomyces pombe. A 361-bp portion of ars3002 containing detectable ARS activity includes multiple near matches to the S. pombe ARS consensus sequence previously reported by Maundrell et al. (K. Maundrell, A. Hutchison, and S. Shall, EMBO J. 7:2203-2209, 1988). Using a gel shift assay with a multimer of an oligonucleotide containing three overlapping matches to the Maundrell ARS consensus sequence, we have detected several proteins in S. pombe crude extracts that bind to the oligonucleotide and ars3002. One of these proteins, ARS binding protein 1, was previously described (Abpl [Y. Murakami, J. A. Huberman, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 93:502-507, 1996]). In this report the isolation, characterization, and cloning of a second binding activity, designated ARS binding protein 2 (Abp2), are described. Purified Abp2 has an apparent molecular mass of 75 kDa. Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consensus sequence. The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coli. The DNA binding activity of overexpressed Abp2 was similar to that of native Abp2. The deduced amino acid sequence contains a region similar to a proline-rich motif (GRP) present in several proteins that bind A+T-rich DNA sequences. Replacement of amino acids within this motif with alanine either abolished or markedly reduced the DNA binding activity of the mutated Abp2 protein, indicating that this motif is essential for the DNA binding activity of Abp2. Disruption of the abp2 gene showed that the gene is not essential for cell viability. However, at elevated temperatures the null mutant was less viable than the wild type and exhibited changes in nuclear morphology. The null mutant entered mitosis with delayed kinetics when DNA replication was blocked with hydroxyurea, and advancement through mitosis led to the loss of cell viability and aberrant formation of septa. The null mutant was also sensitive to UV radiation, suggesting that Abp2 may play a role in regulating the cell cycle response to stress signals.

Amino Acid Sequence↗
PubMed1998

HsMCM2/BM28: a novel proliferation marker for human tumors and normal tissues.

HsMCM2/BM28 is a member of the family of minichromosome maintenance (MCM) proteins, which play a critical role in DNA replication by helping to ensure that DNA is replicated once and only once per cell cycle. The association of HsMCM2 with DNA replication suggested to us that it might prove useful as a new marker for cell proliferation. To test this possibility, we employed immunohistochemistry and immunoblotting to study HsMCM2 expression in both normal human tissues and primary human tumors. We found that HsMCM2 was detectable by immunoblotting in 97% of the studied tumors but in only 27% of the corresponding normal tissues. In normal tissues, the immunoblot signal was weaker than in tumors. Immunohistochemistry revealed that in normal tissues HsMCM2 is present only in proliferating cell nuclei. In most cases, tumor cell nuclei produced a stronger HsMCM2 signal than normal proliferating cell nuclei. Comparative studies revealed that antibodies against HsMCM2 stained fewer nuclei than antibodies against proliferating cell nuclear antigen but usually more than antibodies against Ki-67 (another proliferation-related antigen). Thus, the correlations between proliferation and antigenic signal are different for these three proteins. These results indicate that HsMCM2 is, indeed, a novel marker for proliferating cells. Further studies are required to determine whether the fact that HsMCM2 has a different correlation with proliferation and elevated staining intensity in tumor nuclei (compared to nuclei in normal proliferating cells) will permit it to be a more useful diagnostic and prognostic marker than proliferating cell nuclear antigen and Ki-67.

Biomarkers↗
PubMed1997

Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe.

We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from Schizosaccharomyces pombe. This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA. Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and S. pombe Abp1, and this gene was designated as cbh+ (CENP-B homologue). Cbh protein specifically interacts in vitro with the K-type repeat DNA, which is essential for centromere function. The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides. Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in S. pombe.

Amino Acid Sequence↗
PubMed1997

Mapping replication origins, pause sites, and termini by neutral/alkaline two-dimensional gel electrophoresis.

Neutral/alkaline two-dimensional gel electrophoresis is a robust, easily interpretable, sensitive technique that has yielded insights about the in vivo replication of many types of DNA, from multicopy yeast plasmids to single-copy chromosome regions in unsynchronized mammalian cells. It can provide information about directions of replication fork movement and locations of replication origins, termini, and pause sites. Especially when combined with its partner technique, neutral/neutral two-dimensional gel electrophoresis, it is a method of choice for investigation of unknown situations.

Animals↗
PubMed1997

An origin of replication and a centromere are both needed to establish a replicative plasmid in the yeast Yarrowia lipolytica.

Two DNA fragments displaying ARS activity on plasmids in the yeast Yarrowia lipolytica have previously been cloned and shown to harbor centromeric sequences (P. Fournier, A. Abbas, M. Chasles, B. Kudla, D. M. Ogrydziak, D. Yaver, J.-W. Xuan, A. Peito, A.-M. Ribet, C. Feynerol, F. He, and C. Gaillardin, Proc. Natl. Acad. Sci. USA 90:4912-4916, 1993; and P. Fournier, L. Guyaneux, M. Chasles, and C. Gaillardin, Yeast 7:25-36, 1991). We have used the integration properties of centromeric sequences to show that all Y. lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere. The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere. A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid. The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome. Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not.

Base Sequence↗