Biomedical subjects

D G Kaufman

Publications and source records attributed to D G Kaufman.

At least 19 recordsLinked to original sources

PubMed1992

Partial characterization of nuclear matrix attachment regions from human fibroblast DNA using Alu-polymerase chain reaction.

The proteinaceous nuclear matrix of mammalian cell nuclei has been suggested to be involved in the regulation of chromatin structure, DNA replication, and gene expression. Interaction between cellular DNA and the nuclear matrix is mediated by putative DNA binding sequences, matrix attachment regions (MARs), which may become altered during early events in cellular transformation. Among the cellular changes occurring during the development of neoplasia, all of which may potentially involve the nuclear matrix, are alterations in nuclear structure, loss of control of DNA replication, and significant modifications of cellular gene expression. Therefore, a better understanding of the interaction between DNA and the nuclear matrix is needed. Isolated matrix associated DNA from pulse labeled SV40 transformed human fibroblasts was shown to be enriched in newly replicated DNA, confirming the association of DNA replication with the nuclear matrix as observed by others. Subgenomic fractions of matrix associated DNA enriched in putative MARs sites were prepared from quiescent and logarithmically growing normal human fibroblasts and SV40 transformed human fibroblasts. These fractions of DNA were analyzed by Alu-polymerase chain reaction and agarose gel electrophoresis, revealing complex and unique patterns of DNA products for each cell type investigated. A number of prominent DNA fragments with similar molecular size were found to be present in the amplified DNA products of each DNA source, suggesting that these DNA fragments may represent common DNA sequences which contain MARs sites or which are associated with MARs sites. The application of Alu-polymerase chain reaction to the molecular analysis of nuclear matrix associated DNA may facilitate the isolation and characterization of potentially new human MARs sequences.

DNA↗

PubMed1992

Differential effects of dioctanoylglycerol on fibronectin localization in normal, partially transformed, and malignant human endometrial stromal cells.

In this study, we describe the effects of direct activation of PKC by dioctanoylglycerol (DiC8) on cellular morphology and the localization of fibronectin (Fn) in normal, oncogene-transfected, and malignant human endometrial stromal cells. We questioned whether DiC8, an endogenous specific activator of PKC, would function as a second oncogene in partially transformed human endometrial stromal cells (HESC). Cells utilized were (1) normal HESC, (2) HESC transfected with a plasmid containing an origin-defective temperature-sensitive SV40 large T antigen alone or (3) in combination with an EJ ras oncogene, and (4) an endometrial sarcoma cell line (S7). Cell cultures were treated for 1 h with sn-dioctanoylglycerol (DiC8) and stained with a monoclonal fluorescein-labeled anti-Fn antibody. In normal HESC, DiC8 induced cell rounding and caused Fn localization to revert from the perinuclear region to the cell periphery. All experiments in this investigation were performed when cells were maintained at the permissive temperature for SV40 large T antigen function. In HESC expressing the SV40 large T antigen alone, Fn was localized to the perinuclear region and also occurred as parallel strands between cells. When these cells were treated with DiC8, Fn localization changed to intense punctate regions at the cell periphery or to matrix-like patterns between cells. Also, in these cells, DiC8 induced greater detachment of cells from the substrate than from other cells, resulting in an apparent piling up of cells. Control and treated SV40/EJ ras cells and uterine sarcoma cells expressed Fn in a matrix-like pattern between cells. The rounded cellular morphology of treated HESC and treated cells expressing SV40 resembled the morphology of control or treated SV40/EJ ras cells and uterine sarcoma cells. Thus, treated cells expressing the SV40 large T antigen resembled the SV40/EJ ras cells and uterine sarcoma cells with respect to Fn localization and cellular morphology. DiC8 did not appear to further transform HESC expressing SV40 and EJ ras. However, with regard to cell shape and Fn localization, our results suggest that DiC8 may function as a second oncogene in the signal transduction pathway, in cells expressing SV40 alone. It appears that, with regard to Fn localization, DiC8 may alter signal transduction analogously to that caused by the activated Ha-ras oncogene in HESC expressing the SV40 large T antigen.

Antibodies, Monoclonal↗

PubMed1992

Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.

Antigens, Polyomavirus Transforming↗

PubMed1992

Initiation by bleomycin of hepatocellular foci in the rat.

The antitumor antibiotic, bleomycin, was tested for activity as an initiator of hepatocellular foci and neoplasms in rats. The compound was administered in a single dose via the portal vein 4 h after the proliferative stimulus of a two-thirds partial hepatectomy. Rats were subsequently fed diet containing phenobarbital for up to 41 weeks to promote the development of initiated hepatocytes. Bleomycin-treated livers displayed significantly increased frequencies of basophilic hepatocellular foci and hepatocellular foci which retain glycogen during fasting. Foci that express glutathione-S-transferase (placental form) were not initiated by bleomycin. Hepatocellular neoplasms were infrequently seen in bleomycin-treated livers (5% incidence). The results suggest that oxygen radical-mediated DNA damage may initiate, within populations of proliferating hepatocytes, new lineages of altered hepatocytes that form foci but have low probability of progressing to neoplasms during promotion with phenobarbital.

Animals↗

PubMed1992

Association between expression of transforming growth factor-alpha and progression of hepatocellular foci to neoplasms.

Hepatocarcinogenesis was initiated in rats with a single dose of either of two chemical mutagens--benzo[a]pyrene diolepoxide I and methyl(acetoxymethyl)nitrosamine--administered 15 h after partial hepatectomy. The development of hepatocellular foci and neoplasms was then promoted with dietary phenobarbital given for 45 or 62 weeks. Formalin-fixed tissue specimens that contained hepatic neoplasms and altered hepatocellular foci were screened for expression of the oncodevelopmental marker glutathione-S-transferase (placental form) (GSTP) and transforming growth factor-alpha (TGF-alpha) using immunohistochemistry. All (100%) hepatocellular carcinomas expressed both GSTP and TGF-alpha, as did most hepatocellular adenomas (greater than 80%). However, quantitative stereologic analysis of treated and control livers revealed that GSTP-positive foci were 10-30 times more frequent than TGF-alpha-positive foci. Foci with homogeneous expression of GSTP generally displayed heterogeneous expression of TGF-alpha with reaction product most prominent at their peripheries. Less frequently homogeneous TGF-alpha-positive foci were seen within GSTP-positive foci. The average volumes of those GSTP-positive foci that also expressed TGF-alpha were significantly greater than those of the entire sets of GSTP-positive foci. These results suggest that expression of TGF-alpha may distinguish a subset of GSTP-positive foci that have a growth advantage and increased probability of progression to neoplasia.

Adenoma↗

PubMed1992

Effects of the SV40 large T antigen and EJ ras oncogene on fibronectin localization in human endometrial cells as viewed by confocal laser scanning microscopy.

We utilized confocal laser scanning microscopy to examine the localization of fibronectin deposition in cultures of human endometrial stromal cells. We found that fibronectin in normal human endometrial stromal cell cultures was both intracellular, occurring in rough endoplasmic reticulum and in perinuclear regions, and extracellular, occurring diffusely over the entire cell surface. Endometrial stromal cells were transfected with a plasmid containing an origin-defective Simian Virus 40 (SV40) which codes for a temperature-sensitive large T antigen. When these cells were placed under temperature-restrictive conditions for large T-antigen function, they exhibited staining patterns similar to normal endometrial cells. Fibronectin deposition in cultures of partially or fully transformed endometrial cells was not intracellular as in normal cells, but was localized primarily between cells. Cells expressing the SV40 large T antigen deposited fibronectin mainly in parallel clumps between cells. Cells expressing both the SV40 large T antigen and the EJ ras oncogene, at high cell density, displayed networks of fibronectin arranged in matrix-like patterns between cells. The malignant cell line examined, sarcoma cells, also exhibited fibronectin networks between cells. Cell density affected fibronectin deposition in endometrial stromal cells expressing the EJ ras oncogene. At low density, cells expressing the SV40 large T antigen and the EJ ras oncogene displayed diffuse fibronectin patterns and, at high density, these cells formed colonies with networks of fibronectin between cells.

Antigens, Polyomavirus Transforming↗

PubMed1991

The use of rabbit polyclonal antibodies for the isolation of carcinogen-adducted DNA by immunoprecipitation.

Polyclonal rabbit antibodies elicited against DNA with high levels of (+/-) 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) adducts were used to isolate DNA fragments modified by this carcinogen. DNA treated in vitro with different concentrations of BPDE-I was used as substrate in double-antibody immunoprecipitation reactions. The IgG fraction from immune rabbit serum (primary antibody) was reacted with single-stranded plasmid DNA bearing BPDE-I adducts, and the complexes were immunoprecipitated using goat antirabbit-IgG as secondary antibody. DNA was isolated from the immunoprecipitated pellet, blotted onto nitrocellulose or nylon, and hybridized with 32P-labeled sequences homologous to a fragment of the plasmid DNA used in the assay. The recovery of both DNA and adducts in the immunoprecipitated pellet increased with the level of carcinogen adduction of the DNA. The immunoprecipitation reaction appeared to be more efficient for fragments of DNA containing a high number of adducts. The amount of 32P-hybridizing material recovered by immunoprecipitation was virtually identical to the amount added to the reaction in DNA samples that contained three adducts per 10(3) nucleotides.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1991

Proliferation of carcinogen-damaged hepatocytes during cell-cycle-dependent initiation of hepatocarcinogenesis in the rat.

Hepatocyte proliferation and damage to DNA were characterized during the initiation phase of carcinogenesis in livers of rats that had received a single administration of the methylating agent methyl(acetoxymethyl)nitrosamine (DMN-OAc). Quiescent non-proliferating hepatocytes in intact livers did not appear to be susceptible to initiation by DMN-OAc, whereas proliferating hepatocytes in the S phase appeared to have greatest risk. To characterize the phenomenology of S-phase-dependent initiation further, the fractions of hepatocytes in the S and M phases of the cell cycle were enumerated at various times after treatment with DMN-OAc. Hepatocytes treated when in G1 experienced a delay of up to 20 h in the onset of S phase and a reduced rate of entry into the S and M cycle phases. Hepatocytes treated when in S phase experienced considerable delay in progression to mitosis due to part to inhibition of DNA replication. Hepatocytes treated when in late S/G2 also demonstrated a delay in progression into mitosis. The levels of 7-methylguanine and O6-methyldeoxyguanosine were quantified in the nuclear DNA of proliferating hepatocytes. The kinetics of removal of these lesions appeared to be first-order (half-life = 24 h). Hepatocyte risk of initiation was modeled by a function which summed over time the product of the fraction of hepatocytes in the S phase and the fraction of residual, unrepaired damage to DNA. For hepatocytes treated when in early G1, the time-weighted frequency of premutagenic DNA damage that was present during DNA replication was estimated to be less than half of that for hepatocytes treated when in early S. The results suggest that cell-cycle-dependent variation in sensitivity to initiation of hepatocarcinogenesis may be, in part, due to efficient removal of potentially carcinogenic lesions from DNA during an extended G1. The apparent high sensitivity of hepatocytes in late S/G2 suggests the contribution of additional factors.

Animals↗

PubMed1991

Extended life span of human endometrial stromal cells transfected with cloned origin-defective, temperature-sensitive simian virus 40.

Human endometrial stromal cells transfected with an origin-defective, temperature-sensitive simian virus 40 recombinant plasmid are dependent on T-antigen function for proliferation and at the permissive temperature have an extended life span in culture. Southern blot analysis indicates that the transfected gene is present in low copy number, possibly at a single integration site. Normal stromal cells are capable of 10 to 20 population doublings in culture. Transfected cultures have been carried at the permissive temperature to 80 population doublings before crisis. In the multistep model of malignant transformation of human cells, these cells represent one of the earliest stages: extended but finite life span. We have used these cells to investigate alterations in signal transduction that may be responsible for this early stage of transformation caused by the large T antigen. Temperature shift experiments indicate that the expression of ornithine decarboxylase (ODC) but not of c-fos is altered by the large T antigen. Induction of c-fos by serum or 12-O-tetradecanoylphorbol-13-acetate is independent of temperature. However, in the transfected cells, the induction of ODC by asparagine or serum is greatly enhanced at the permissive temperature. This result indicates that the large T antigen acts downstream of c-fos but upstream of ODC expression in the signal-transducing cascade.

Antigens, Polyomavirus Transforming↗

PubMed1991

Fluorescent laser scanning microscopy of F-actin disruption in human endometrial stromal cells expressing the SV40 large T antigen and the EJ ras oncogene.

To attempt to understand the effects of the SV40 large T antigen and an activated EJ ras oncogene on F-actin organization, we compared normal human endometrial stromal cells (HESC; proliferating, short life span) to cells transfected with the SV40 large T antigen either alone or in combination with the EJ ras oncogene. Normal HESC displayed numerous bundles of actin filaments (stress fibers) evenly distributed throughout the cell. In HESC transfected with a plasmid containing the gene for a temperature-sensitive SV40 large T antigen, stress fibers were disrupted and the remaining F-actin was also disrupted and clumped near the plasma membrane. Cells expressing both the SV40 large T antigen and the EJ ras oncogene sometimes appeared rounded, with stress fibers organized mainly near the cell periphery. Under restrictive temperature conditions for the function of the SV40 large T antigen, cells with or without the EJ ras oncogene reorganize actin stress fibers to resemble those of normal HESC. Therefore, the EJ ras oncogene alone does not disrupt F-actin organization. When operating in cooperation with the SV40 large T antigen, however, it leads to the reorganization of F-actin at the cell periphery and confers a rounded structure on the cells.

Actins↗

PubMed1991

Automated image-based cytometry with fluorescence-stained specimens.

The combination of digitized microscopy, algorithms for object recognition and fluorescent labeling is a promising approach for reliable, quick, automated and cost-effective screening of clinical specimens. We describe two conceptually different algorithms for detecting objects in fluorescence microscopic images. One, which is partially automated, compares a mask that represents a typical object with every position in the image; the other, which is fully automated, calculates threshold intensities to segment the image into regions of objects and background. Applications of the algorithms in conjunction with a prototype image-based cytometer are demonstrated for determining the DNA ploidy distribution of cultured human endometrial cells and determining the DNA ploidy distribution and the fraction of cells expressing the E6 antigen of human papilloma virus serotypes 16 and 18 in a PAP smear. The encouraging results from this study suggest that automated image-based cytometry utilizing fluorescent stains will be a valuable asset for clinical screening.

Algorithms↗

PubMed1991

Automated fluorescence image cytometry. DNA quantification and detection of chlamydial infections.

Digitized fluorescence microscopy in conjunction with automated image segmentation is a promising approach for screening clinical specimens quickly and reliably. This paper describes the hardware and software of a prototype image-based cytometer that can identify fluorescent objects, discriminate true objects from artifacts and divide overlapping pairs of objects. The use of this image cytometer is discussed for: (1) the measurement of the DNA ploidy distribution of isolated mature rat liver nuclei labeled with 4',6-diamidine-2-phenylindole; (2) the comparison of the DNA ploidy distributions of the same samples measured by image cytometry (ICM) and flow cytometry (FCM); and (3) the quantification of chlamydial infection by double labeling cells with antichlamydiae antibody and Hoechst 33258 for nuclear DNA analysis. Ploidy distributions measured by the automated image cytometer compared favorably to those obtained by FCM. All pairs of overlapping nuclei were automatically detected by an additional computer algorithm, and those pairs that were clearly more than one nucleus by visual inspection were correctly divided. The irregular morphology of the chlamydiae-infected cells meant that 26% of them were not correctly identified in the fluorescein-stained images (as judged by manual inspection), but all cells were nevertheless detected correctly from the images of the Hoechst-stained samples. Automated fluorescence ICM yielded results similar to those obtained with FCM and had the additional benefit of maintaining cell and tissue architecture while preserving the opportunity for subsequent manual inspection of the specimen.

Algorithms↗

PubMed1991

A herald bleed: a case of aortoesophageal fistula and a review of the literature.

Survival in AEF is rare because the diagnosis of this uncommon entity is not always suspected, and few patients survive despite aggressive but often late intervention. We present this case to increase awareness of AEF, which although rare, does occur and should be suspected in any patient who presents with midthoracic pain or dysphagia and herald bleeding.

Aged↗

PubMed1990

Expression of transforming growth factor beta 1 by human endometrial carcinoma cell lines: inverse correlation with effects on growth rate and morphology.

We examined the effects of transforming growth factor beta 1 (TGF-beta 1) on various aspects of the cell biology of human endometrial carcinoma (HEC) cell lines in vitro, as well as the expression of TGF-beta 1 mRNA by these cell lines. Cell lines from eight HEC tumors, representing a variety of histological subtypes, were studied in order to test the generality of conclusions regarding the effects of TGF-beta 1 on this particular tumor cell type. The growth of five HEC cell lines was inhibited by TGF-beta 1 (10 ng/ml), while growth of three cell lines was not inhibited. The effects on growth correlated with morphological alterations induced by TGF-beta 1; the cell lines with inhibited growth displayed a larger, flatter, more contact-inhibited phenotype, while the cell lines whose growth ws not inhibited showed few discernible morphological alterations in response to TGF-beta 1. Northern analysis of TGF-beta 1 mRNA levels revealed that the three HEC cell lines unresponsive to TGF-beta 1 treatment expressed relatively large amounts of TGF-beta 1. Correspondingly, the five HEC cell lines which responded to TGF-beta 1 with growth and morphological changes expressed much lower levels of TGF-beta 1 mRNA. These results suggest that the sensitivity of human HEC cell lines to TGF-beta 1 is variable and that this sensitivity is inversely correlated with the level of expression of TGF-beta 1.

Carcinoma↗

PubMed1990

Ultrastructural characterization of two new human endometrial carcinoma cell lines and normal human endometrial epithelial cells cultured on extracellular matrix.

Two new lines of human endometrial carcinoma (HEC) cells, one from an adenocarcinoma and one from a highly metastatic serous papillary carcinoma, were established in culture. Structural and morphologic properties of these cells at early passage were compared with those of cultured normal human endometrial epithelial (NHEE) cells. For these studies, cells were grown on a conventional plastic surface or on an extracellular matrix substrate (Matrigel), and examined by transmission electron microscopy and immunofluorescent light microscopy. The HEC cells appeared morphologically similar on plastic and Matrigel, whereas the NHEE cells showed significantly greater epithelial morphologic differentiation on Matrigel than on plastic. On extracellular matrix, the morphologic differences observed between HEC cells and NHEE cells were primarily of an architectural nature, which may be in part explained by differences between NHEE and HEC cells in the arrangement of actin microfilaments and cytokeratin intermediate filaments. Furthermore, HEC cells displayed extensive networks of vimentin intermediate filaments, which were absent from the NHEE cells. These observations support the hypothesis that architectural deregulation is a prominent feature of endometrial carcinoma, and that cytoskeletal alterations may uncouple HEC cell ultrastructural morphology from the influence of extracellular matrix.

Actin Cytoskeleton↗

PubMed1990

Carcinoma in situ of the testes: diagnosis by aspiration flow cytometry.

Carcinoma in situ of the testes has been described as a premalignant state with the potential to progress to invasive carcinoma. A history of testicular carcinoma, cryptorchidism, somatosexual ambiguity and infertility has been identified as a risk factor for carcinoma in situ. A series of 25 infertility patients underwent aspiration biopsy of the testis as part of a study protocol to assess spermatogenesis. Of these patients 1 had a unilateral seminoma. In accordance with the study protocol the contralateral testis was aspirated at radical orchiectomy and the deoxyribonucleic acid histogram generated demonstrated an aneuploid peak. Carcinoma in situ subsequently was noted on tissue biopsy. The discovery of carcinoma in situ cells in this manner highlights the potential of this technique as a diagnostic tool for the screening and followup of men at increased risk for carcinoma in situ.

Adult↗

PubMed1990

Malignant transformation of human endometrial stromal cells by transfection of c-myc: effects of pRSVneo cotransfection and treatment with MNNG.

Stromal cells isolated from normal human endometrium were cotransfected in primary culture with pSVc-myc, a plasmid containing a truncated c-myc gene regulated by simian virus 40 promoter, and pRSV neo, a plasmid containing a neomycin resistance gene regulated by Rous sarcoma virus (RSV) promoter. These cells demonstrated properties of transformed cells in vitro, including altered morphology, focus formation, anchorage-independent growth, chromosomal alterations, and tumor formation in athymic mice. When these cells were treated subsequently with a direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine, they demonstrated higher colony-forming efficiency in soft agar and reduced tumor latency. Cells transfected with pRSV neo alone exhibited some properties associated with neoplastic transformation, including altered morphology and formation of colonies in soft agar. It is presumed that in normal cells transfected with pRSV neo, RSV long terminal repeats activated cellular genes that normally regulate growth of human endometrial stromal cells.

Avian Sarcoma Viruses↗

PubMed1989

Establishment and characterization of a human cell line from a serous papillary endometrial carcinoma.

The cell line SPEC-1, derived from a human serous papillary endometrial carcinoma (SPEC), has been established and repetitively subcultured for over 18 months. SPEC is a clinically aggressive histologic variant of endometrial adenocarcinoma with a significantly poorer prognosis. The SPEC cells exhibit morphologic and ultrastructural characteristics of transformed epithelial cells. The cells were further characterized with regard to growth kinetics, histochemistry, karyotype, and tumorigenicity. These studies indicate that several properties of the SPEC cells in culture contrast markedly with those of both typical endometrial adenocarcinoma cell lines and normal endometrial epithelia, as described in the literature. The most significant of these differences concern cytogenetic and ultrastructural features. The implications of these unique characteristics are discussed with regard to the relationship they may have to the unusually aggressive biological behavior of this tumor cell type in vivo. This SPEC cell line should prove useful in future studies designed to determine important factors in the biological behavior of human tumor cells.

Animals↗