Search PubMed⌕ Search

Biomedical subjects

J D Knox

Publications and source records attributed to J D Knox.

At least 19 recordsLinked to original sources
PubMed2000

Inhibition of DNA methyltransferase inhibits DNA replication.

Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.

Animals↗
PubMed2000

DNA methyltransferase inhibition induces the transcription of the tumor suppressor p21(WAF1/CIP1/sdi1).

Previous lines of evidence have shown that inhibition of DNA methyltransferase (MeTase) can arrest tumor cell growth; however, the mechanisms involved were not clear. In this manuscript we show that out of 16 known tumor suppressors and cell cycle regulators, the cyclin-dependent kinase inhibitor p21 is the only tumor suppressor induced in the human lung cancer cell line, A549, following inhibition of DNA MeTase by a novel DNA MeTase antagonist or antisense oligonucleotides. The rapid induction of p21 expression points to a mechanism that does not involve demethylation of p21 promoter. Consistent with this hypothesis, we show that part of the CpG island upstream of the endogenous p21 gene is unmethylated and that the expression of unmethylated p21 promoter luciferase reporter constructs is induced following inhibition of DNA MeTase. These results are consistent with the hypothesis that the level of DNA MeTase in a cell can control the expression of a nodal tumor suppressor by a mechanism that does not involve DNA methylation.

Cell Division↗
PubMed1999

Identification of initiation sites for DNA replication in the human dnmt1 (DNA-methyltransferase) locus.

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.

Chromosome Mapping↗
PubMed1999

Modified oligonucleotides as bona fide antagonists of proteins interacting with DNA. Hairpin antagonists of the human DNA methyltransferase.

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. Ex vivo, hairpin inhibitors of DNA MeTase are localized to the cell nucleus in lung cancer cells. They inhibit DNA MeTase, cell growth, and anchorage independent growth (an indicator of tumorigenesis in cell culture) in a dose-dependent manner. The inhibitors developed in this study are the first documented example of direct inhibitors of DNA MeTase in living cells and of modified oligonucleotides as bona fide antagonists of critical cellular proteins.

Base Sequence↗
PubMed1998

Synthetic matrix metalloproteinase inhibitor, BB-94, inhibits the invasion of neoplastic human prostate cells in a mouse model.

BACKGROUND: It has been suggested that increased metalloproteinase activity is a critical event in neoplastic progression leading to the initiation of local invasion and ultimately to the dissemination of neoplastic cells. This has led to an interest in testing the ability of metalloproteinase inhibitors to prevent the progression of carcinoma in situ into invasive and, therefore, more malignant tumors. One such agent is the synthetic matrix metalloproteinase inhibitor, BB-94. METHODS: The effect of BB-94 on the intrinsic invasive potential of matrilysin-transfected Du-145 cells was evaluated by an in vitro invasion assay. In addition a diaphragm invasion model, which provides an easily oriented structure in which the earliest penetration of the basal lamina can be observed, was used to investigate the effect of BB-94 on the invasion and growth of tumors formed by these cells when injected into S.C.I.D. mice. RESULTS: The synthetic matrix metalloproteinase inhibitor, BB-94, was shown to effectively inhibit the invasion of matrigel and murine diaphragm. CONCLUSIONS: Metalloproteinase inhibitors, such as BB-94, that are able to limit tumor growth, and local invasion, may decrease the invasion of invasive carcinomas.

Adenocarcinoma↗
PubMed1998

Concurrent replication and methylation at mammalian origins of replication.

Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.

Animals↗
PubMed1996

Matrilysin expression in human prostate carcinoma.

The metalloproteinases, a multigene family of metal-requiring enzymes, have been suggested to play a role in tumor invasion and metastasis. Previously, we demonstrated that human primary prostate tumors express higher levels of matrilysin and gelatinase A mRNA than normal prostate does. In the study presented here, we used in situ hybridization and immunohistochemical staining of serial sections of paraffin-embedded primary prostate tumors to compare the sites of matrilysin and gelatinase A expression and protein localization. These results confirmed the epithelial nature of matrilysin expression and protein localization. In contrast, gelatinase A mRNA was localized to the interstitial stroma, whereas the protein was associated with the epithelial tumor cells. In situ hybridization was also used to demonstrate that gelatinase B expression was restricted to macrophages infiltrating the tumors. Proteins isolated from an additional set of frozen tumor specimens were analyzed by western blotting to determine the relative amounts of matrilysin in the active and proenzyme forms. The western analyses demonstrated that in all cases in which matrilysin was detected, at least some of the enzyme was in the active form. These results are discussed with respect to the possible role these enzymes may play in prostate tumor progression.

Amino Acid Sequence↗
PubMed1996

Mapping of the metalloproteinase gene matrilysin (MMP7) to human chromosome 11q21-->q22.

The matrix metalloproteinase, matrilysin, is thought to play an important role in the early steps of tumor progression. We determined the chromosome location of the matrilysin gene (MMP7) by Southern and PCR analysis of two different panels of somatic cell hybrids and in situ hybridization of metaphase chromosomes. Matrilysin maps to the region, 11q21-->q22, adding MMP7 to the cluster of matrix metalloproteinase genes that have already mapped to this region.

Base Sequence↗
PubMed1995

Expression of hemidesmosomal and extracellular matrix proteins by normal and malignant human prostate tissue.

The progression of prostate carcinoma may be influenced by the biochemical nature of the basal lamina surrounding the primary carcinoma cells. As a first step toward understanding this process, the composition and structure of the basal lamina in normal prostate, prostatic intraepithelial neoplasia, and human carcinoma were determined. In addition, a comparison was made between the attachments of the normal basal cell to its underlying basal lamina and those made by primary prostate carcinoma. The normal basal cells form both focal adhesions and hemidesmosomal-like structures as observed by transmission electron microscopy. The normal basal cells exhibited a polarized distribution of hemidesmosomal associated proteins including BP180, BP230, HD1, plectin, laminin-gamma 2(B2t), collagen VII, and the corresponding integrin laminin receptors alpha 6 beta 1 and alpha 6 beta 4. The expression and distribution pattern of these proteins were retained in the prostate intraepithelial neoplasia lesions. In contrast, the carcinoma cells uniformly lacked hemidesmosomal structures, the integrin alpha 6 beta 4, BP180, laminin-gamma 2 (B2t), and collagen VII but did express BP230 (30%), plectin, HD1 (15%), and the integrin laminin receptors alpha 3 beta 1 and alpha 6 beta 1. These results suggest that, although a detectable basal lamina structure is present in carcinoma, its composition and cellular attachments are abnormal. The loss of critical cellular attachments may play a role in influencing the progression potential of prostate carcinoma.

Carcinoma↗
PubMed1994

Adhesion molecules, extracellular matrix, and proteases in prostate carcinoma.

Immunohistochemical studies of prostate carcinoma reveal that most primary carcinomas, including high-grade tumors, are surrounded by a basal lamina composed of laminin, type IV collagen, and entactin. In addition to the expected laminin subchains A, B1, B2, subchains M and S are also found. Tenascin, found around normal glands, is seen in 60% of carcinomas. The basal cells of the normal gland express several integrin alpha units including alpha 2,3,4,5,6, and v. Both beta 1 and beta 4 subunits are observed. These integrin units are polarized at the base of the cells where they codistribute with the surrounding extracellular matrix. The integrin alpha 6 beta 4 is associated with hemidesmosomal-like structures, as detected by transmission electron microscopy (TEM). In carcinoma, the beta 4 is not observed and the alpha 6 and beta 1 subunits are variably expressed. The integrin expression in carcinoma is diffuse in the cytoplasmic membrane and not restricted to the basal aspects of the cell. In addition, type VII collagen and the BP 180 protein which are associated with hemidesmosomes are lost, although the BP 230, plectin, and HD1 proteins are variably expressed. Using immunohistochemistry and northern analysis we observed three metalloproteinases in prostate carcinoma--matrilysin, gelatinase A, and gelatinase B. Western blotting and zymogram analysis reveal that of these three, only matrilysin appears to be present in its active form. Recent in situ hybridization studies reveal focal expression of the matrilysin mRNA in 25/33 primary carcinomas. Matrilysin also appears to be highly expressed in prostatic ducts and atrophic glands. Expression of the three metalloproteinases is also seen in prostatic intraepithelial neoplasia lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Autopsy↗
PubMed1994

Differential expression of extracellular matrix molecules and the alpha 6-integrins in the normal and neoplastic prostate.

The epithelial basal lamina composition and integrin expression profile of normal and neoplastic human prostate was characterized using immunohistochemical analysis of frozen samples. The major components of the basal lamina surrounding normal acini were laminin, type IV collagen, entactin, and type VII collagen with variable amounts of tenascin. The basal lamina of neoplastic acini had a similar composition, except for the loss of type VII collagen, which was observed in all grades of carcinoma. The basal cells of the normal prostate express the alpha 6-, beta 1-, and beta 4-integrin subunits, suggesting that both the alpha 6 beta 1- and alpha 6 beta 4-integrin complexes are formed. In prostate carcinoma there is a complete loss of beta 4 expression and the alpha 6- and beta 1-integrin subunits, which are restricted to the basal and basal lateral surfaces of basal cells, are distributed diffusely throughout the cytoplasmic membrane. The differential expression of type VII collagen and beta 4 are discussed in relationship to their possible role in tumor progression.

Aged↗
PubMed1993

Expression of the metalloproteinase matrilysin in DU-145 cells increases their invasive potential in severe combined immunodeficient mice.

Human prostate cancer displays a high degree of variability in its rate of spread, which could be due largely to differences in the invasive potential of the tumor cells. The degradation of the basal lamina and stromal extracellular matrix is mediated in part by the secretion of matrix metalloproteinases (MMPs). Matrilysin (PUMP-1, MMP-7) and gelatinase A (M(r) 72,000 type IV collagenase, MMP-2) have been shown to be overexpressed in prostate carcinoma. We have expressed the single MMP matrilysin in the tumorigenic but nonmetastatic human prostate tumor cell line DU-145 to determine if matrilysin has a functional role in prostate tumor cell invasion. DU-145 cells expressing matrilysin were significantly more invasive than vector-only transfected cell lines as assayed by a severe combined immunodeficient mouse model of tumor cell invasion. Vector-only transfected DU-145 cells injected i.p. into severe combined immunodeficient mice invaded the diaphragm in only 1 of 9 mice (11%), whereas matrilysin-transfected DU-145 cells invaded the diaphragm in 12 of 18 mice (66%). The difference between the controls and matrilysin-transfected cells was statistically significant (P < 0.006). These results suggest a functional role for matrilysin in the initial invasion of prostate cancer through the epithelial basal lamina and into the surrounding stroma.

Animals↗
PubMed1993

Effects of taxol and taxol/hyperthermia treatments on the functional polarization of cytotoxic T lymphocytes.

Immunofluorescence staining, electron microscopy, and (51Cr) cytolytic release assays are used to investigate the effects of taxol and taxol/hyperthermia treatments on the microtubule organization and cytolytic activity of cytotoxic T lymphocytes (CTLs). A 4 h treatment of CTLs with 1 microM taxol results in an extensive reorganization of the microtubule system to form one to a few large microtubule bundles that extend from the centrosome. The Golgi apparatus is not disrupted by this treatment and remains associated with the microtubule organizing centre (MTOC). This microtubule reorganization has no effect on the ability of CTLs to orient their MTOC towards a bound target cell, nor on their cytolytic activity. In control CTLs, not treated with taxol, a mild hyperthermia treatment (42 degrees C, 30 min) results in an aggregation of the pericentriolar material, a loss of MTOC orientation, an inhibition of cytolytic activity, and a disorganization of the microtubule system [Knox et al.: Exp. Cell Res. 194:275-283, 1991]. In contrast, in taxol-treated CTLs the stabilized microtubule bundles are unaffected by such hyperthermia treatment; however, the other effects of hyperthermia appear identical in control and taxol-treated CTLs. These results indicate that a dynamic, radially arranged microtubule array is not required for the functional polarization of CTLs and suggest that a component of the pericentriolar material may play a key role in effecting MTOC orientation.

Animals↗
PubMed1993

Prostate tumor cell invasion: a comparison of orthotopic and ectopic models.

Interest in orthotopic models has been generated by recent reports of increased invasive and metastatic potential demonstrated by tumor cell lines following injection into their tissue of origin rather than subcutaneously. We have previously demonstrated that transfection of the tumorigenic human prostate cell line, Du-145, with the metalloproteinase matrilysin increased its ability to invade the diaphragm following an intraperitoneal injection. In this study we compare the invasive and metastatic behavior of transfected Du-145 cell lines injected into the dorsal lateral lobe of the prostate to that observed when they are injected intraperitoneally. Immunohistochemistry was used to examine 37 orthotopically injected severe combined immunodeficient mice for local invasion and metastatic lesions. In addition, the effect of injection site on the level of expression of four genes thought to influence the invasiveness of tumor cells (matrilysin, stromelysin, TIMP-1, and TIMP-2), was determined by northern analysis of orthotopic and subcutaneous tumor tissue. The results demonstrate that the level of mRNA expression of the genes examined was similar at the two sites of injection and that the invasive properties of Du-145 cells following orthotopic implantation were comparable to that observed on the diaphragm following intraperitoneal injection. The advantages of the diaphragm invasion model are: less procedure-related mortality, ease of cell delivery, and provision of an easily orientated structure in which the earliest penetration of a basal lamina can be observed.

Adenocarcinoma↗
PubMed1991

Effects of hyperthermia on microtubule organization and cytolytic activity of murine cytotoxic T lymphocytes.

When murine cytotoxic T lymphocytes (CTL) are heated at 42 degrees C for 30 min their ability to lyse their target cells (TC) is severely impaired. When the CTL are allowed to recover at 37 degrees C, a partial recovery of cytolytic activity that peaks within 6 h is observed. A dye exclusion assay demonstrated that such a heat shock does not affect the viability of the CTL and direct microscopic observations established that their ability to bind to TC is not impaired. Therefore, the step or steps inhibited by hyperthermia are subsequent to TC recognition and binding. Kupfer et al. ((1983) Proc. Natl. Acad. Sci. USA 80, 7224-7228) demonstrated that upon binding to an appropriate TC, a rapid orientation of the Golgi apparatus and the microtubule organizing center (MTOC) occurred within the CTL so that the two organelles face the TC. This orientation is a prerequisite for efficient TC lysis. We have shown by immunofluorescence and confocal microscopy, using a monoclonal antibody to tubulin and a rabbit autoimmune serum that binds a centriole-associated protein, that the organization of the MTOC-microtubule array is disrupted by hyperthermia. EM suggests that this disorganization of the microtubules may result from an aggregation of the pericentriolar material. The recovery of cytolytic activity is coincident with the reorganization of the microtubules about the MTOC. These findings suggest that the initial inhibitory effect of hyperthermia on CTL function results from the disruption of microtubule organization.

Animals↗
PubMed1989

Biologic variation of common hematologic laboratory quantities in the elderly.

Analytic, within-subject, and between-subject biologic variations were estimated for leukocytes, erythrocytes, hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin content (MCHC), platelets, and a three-component differential count (lymphocytes, monocytes, and granulocytes in terms of both concentration and percentage of leukocytes) in cohorts of 12 male and 12 female healthy elderly subjects. The assays were performed with an Ortho ELT-800 automated analyzer. The estimates of within-subject biologic variation were similar to published data on young subjects, indicating that this aspect of homeostasis is not compromised in the elderly. The data were used to derive objective analytic goals; goals were surpassed except for assays of erythrocytes, hematocrit, and the derived MCV, MCH, and MCHC. The changes required for serial results to be significantly different were determined and found to be generally valid because most quantities have no heterogeneity of within-subject variation. All quantities had significant individuality; in consequence, conventional population-based reference values are of limited utility, and screening using reference limits will not detect latent or early disease in many subjects.

Aged↗
PubMed1989

Breaking bad news: medical undergraduate communication skills teaching and learning.

Teaching on the theme of 'breaking bad news' was selected for special development as part of an introductory course on communication skills for preclinical medial students in 1987. Eight parents of handicapped children and three patients terminally ill with neoplastic diseases cooperated in a series of semi-structured group discussions in which students were encouraged to interact with patients and children. The patients/parents readily assumed the role of teachers and where it was possible, they participated in similar sessions the following year. Analysis of questionnaires indicated that the plan had proved acceptable to patients, relatives and students. Follow-up of student opinion one year later indicated that students felt they had derived benefit from these opportunities to learn first-hand from patients and parents of handicapped children.

Adult↗