Biomedical subjects

D G Kaufman

Publications and source records attributed to D G Kaufman.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1989

Ferritin-labeled rabbit Fab fragments for the single-step detection of benzo[a]pyrene-diol-epoxide adducts in DNA by electron microscopy.

In this paper we describe an immunological method for the visualization of (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-I) adducts in DNA by electron microscopy (EM). The immunoglobulin fraction of rabbit antiserum specific for BPDE-I adducts was digested with papain, the Fab fragments were purified by affinity chromatography on protein A-Sepharose and cross-linked to ferritin. The reactivity of the Fab fragments coupled to ferritin was determined by using anti-ferritin antibodies to precipitate the complexes formed between ferritin-labeled Fab fragments and BPDE-I-modified DNA that had been uniformly labeled with [14C]thymidine. DNA from cells treated with BPDE-I in culture was reacted with ferritin-labeled Fab fragments, separated from unreacted Fab using a Sepharose CL-4B column, and examined by EM. An aliquot of the same DNA was used to determine the level of BPDE-I adduction using an enzyme-linked immunosorbent assay (ELISA). Close agreement was found between the levels of adduction determined by ELISA and EM. A good correlation was also found between the level of adduction measured by EM and scintillation spectrometry when DNA was modified with [3H]BPDE-I in vitro. The EM method presents the following advantages: (i) it avoids cross-linking of separate adducts by the same IgG molecule; and (ii) it requires only one antigen-antibody reaction and a single purification step, allowing analysis of very small amounts of DNA.

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

PubMed1988

Timing of proto-oncogene replication: a possible determinant of early S phase sensitivity of C3H 10T1/2 cells to transformation by chemical carcinogens.

The temporal order of replication of several genes was studied in 10T1/2 cells synchronized by release from confluence-induced arrest of proliferation followed by treatment with 2 micrograms/mL aphidicolin for 24 h. DNA subjected to bromodeoxyuridine substitution for 1- or 2-h intervals spanning the S phase was separated from the remaining DNA in cesium chloride gradients, filtered onto nitrocellulose in a slot-blot apparatus, and hybridized with various 32P-labeled probes. Ha-ras was among the first genes replicated at the onset of the S phase. The myc proto-oncogene replicated later but within the first hour of the S phase. The replication of Ki-ras, raf, and mos was detected between hour 1 and 2 of the S phase. The dihydrofolate reductase gene replicated early (0-2 h) and the myb proto-oncogene replicated in mid-S phase (2-4 h). An immunoglobulin VH sequence and the beta-globin gene replicated late in 10T1/2 cells, 4-6 h after removal of aphidicolin. Replicating DNA is preferentially adducted by chemical carcinogens, and replication of damaged proto-oncogenes before they are repaired may activate their transforming potential. Therefore, the observed replication of proto-oncogenes during the early S phase may underlie the enhanced sensitivity of 10T1/2 cells to chemically induced transformation at this point in the cell cycle.

Animals↗

PubMed1988

Gland formation from human endometrial epithelial cells in vitro.

We have developed methods for the culture of human endometrial glandular epithelia in vitro. The culture medium is serum-free and is used in combination with Matrigel, an extracellular matrix material applied as a coating on cell culture plates. Cell growth begins as a monolayer, but the cells subsequently form glandular or organoid structures. The glands are composed of polar columnar cells facing a central lumen, which is enclosed by the apical surfaces of cells displaying numerous microvilli and sealed by tight junction complexes. The ability to study in vitro the complex process of glandular morphogenesis represents an important new tool in cell biology which may be used to investigate growth regulation, hormone production and dependency, and cellular recognition and interactions. Ultimately, these characteristics may be applied to study the alterations of glandular epithelia associated with neoplasia.

Cells, Cultured↗

PubMed1988

Benzo[alpha]pyrene diol epoxide I binds to DNA at replication forks.

The distribution of lesions in DNA caused by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [alpha]pyrene (B[alpha]P diol epoxide-I) was studied in synchronized C3H/10T1/2 cells treated in S phase. Sites of carcinogen modification of DNA were identified by polyclonal rabbit antibodies elicited against DNA modified with B[alpha]P diol epoxide-I in vitro. This antigenic DNA contained trans-(7R)-N2-[10-(7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene)-yl]- deoxyguanosine; other adducts were not detected by liquid chromatography. In this study, DNA replication forks with antibodies bound to B[alpha]P diol epoxide-I adducts were detected by electron microscopy. The frequency of replication forks containing carcinogen adducts associated with the fork junction was found to be 8-fold higher than expected for an average distribution. The proportion of replication forks that were apparently blocked at the site of the DNA damage increased when replication was allowed to occur after carcinogen exposure. These results support the conclusions that the fork junction is particularly vulnerable to adduction by B[alpha]P diol epoxide-I and that B[alpha]P diol epoxide-I adducts block the displacement of replication forks during DNA synthesis in intact cells.

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

PubMed1988

Phenobarbital-dependent proliferation of putative initiated rat hepatocytes.

The mitogenic effects of phenobarbital (PB) were examined using cultures of putative initiated hepatocytes that proliferate and form colonies under conditions in which normal hepatocytes senesce and die. The frequencies of colony-forming hepatocytes in primary cultures isolated 2 weeks after initiation with methyl(acetoxymethyl)nitrosamine or benzo[a]pyrene-7,8-diol-9,10-epoxide(anti) were in the range of 2-38 per million in the presence of PB. Colony-formation frequencies were 0.1 per million in the absence of PB. Proliferative hepatocyte colonies were not observed in cultures grown in serum-free medium containing PB, epidermal growth factor, nor-epinephrine and insulin. The requirement for PB was characterized further using secondary cultures of hepatocytes that had been isolated from a liver 5 weeks after initiation. The colony-forming efficiency of these hepatocytes was about 10% in the presence of 2 mM PB and less than 0.2% in its absence. Colony formation displayed a linear response to concentrations of PB in the range of 0.5-2 mM and a decline above the optimal 2 mM concentration. Autoradiography was used to determine the percentages of hepatocytes in secondary cultures that synthesized DNA in the presence or absence of PB. By the third day after seeding as single cells, hepatocytes exhibited a labeling index of about 50% and this level of labeling was preserved for up to 2 weeks after seeding. Very few hepatocytes were found to synthesize DNA in the absence of PB and most senesced. A small fraction of the colony-forming hepatocytes continued to proliferate in the absence of PB and formed colonies with a high labeling index. These results suggest that the proliferation of initiated hepatocytes in vivo may be conditional upon the presence of the hepatic tumor promoter, PB.

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

PubMed1987

Cell cycle-dependent initiation of hepatocarcinogenesis in rats by (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)py rene.

Male F344 rats were treated once with (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)pyre ne (BPDE) at various times after a two-thirds partial hepatectomy. Diet containing 0.05% phenobarbital was fed subsequently to promote the development of hepatocellular neoplasms from initiated cells. This combined treatment caused an apparently continuous emergence of hepatocellular neoplasms between 31 and 60 weeks after initiation. Yields of hepatocellular neoplasms (adenomas and carcinomas) seen at 45 weeks after initiation varied according to the time of treatment with BPDE. Rats treated at 15 h after partial hepatectomy, when maximal fractions of proliferating hepatocytes were entering the S phase, displayed the greatest incidence and yield of hepatocellular neoplasms. Rats treated when hepatocytes were early in the prereplicative G1 phase demonstrated a significantly lower incidence and yield of hepatocellular neoplasms. No neoplasms were seen in livers of rats treated with BPDE without prior partial hepatectomy; this indicates the insensitivity or resistance of nonproliferating G0 hepatocytes to initiation by this chemical. The variation in yields of neoplasms could not be attributed to variation in binding of carcinogen to DNA or to rates of DNA repair. The risk of initiation of hepatocytes appeared to be correlated (r = 0.95) with the fraction of hepatocytes that were entering the S phase at the time of treatment with BPDE. These results reveal a significant cell cycle dependency for initiation of hepatocarcinogenesis by BPDE and show that proliferating hepatocytes are at greatest risk when early in the S phase. Quantitation of islands of hepatocellular alteration suggested a similar pattern of cell cycle-dependent sensitivity, although the cycle-dependent variation in island frequencies (1.4-fold) was less than the variation in yields of neoplasms (5-fold). However, 1300-5000 islands were estimated to appear for every one neoplasm that appeared. When viewed within the context of multistep carcinogenesis, and assuming that islands of altered hepatocytes represent a step in this process, then these results suggest that islands may represent populations of cells at least one step removed from neoplasia.

Animals↗

PubMed1987

Flow cytometric analysis of human uterine sarcomas and cell lines.

Flow cytometric techniques were used to characterize multiple human uterine sarcomas and cell lines derived from some of these tumors. Analysis of DNA content showed that 9 of the 11 uterine sarcomas investigated were composed of at least one aneuploid population as well as a distinct diploid population. These data indicate that aneuploidy, as measured by flow cytometry, is a characteristic more common to uterine sarcomas than that previously reported for uterine adenocarcinomas. Unlike the original tumors, the cell lines established from three of the sarcomas contained predominantly diploid populations with only minor aneuploid populations. Treatment of one of the sarcoma cultures with tumor promoters did not result in an increase in the aneuploid populations. Tumors which arose in nude mice upon transplantation of two of the sarcomas did not contain the same distribution of tumor subpopulations as found in the original sarcomas. Apparently, the in vitro culture and and in vivo nude mouse conditions were not appropriate for maintaining the original equilibrium between the aneuploid and diploid subpopulations but instead provided a selective environment that resulted in the preferential growth of only certain tumor populations. Dual-parameter analysis of DNA content and alkaline phosphatase levels of one of the sarcomas were useful for distinguishing the aneuploid from the diploid population coexisting in this tumor. Our data suggest that flow cytometry is a valuable tool to analyze the characteristics of the tumor populations residing in primary uterine sarcomas as well as to determine which of these tumor subpopulations survive in culture and transplantation to nude mice.

Alkaline Phosphatase↗

PubMed1987

Cell cycle-dependent initiation of hepatocarcinogenesis in rats by methyl(acetoxymethyl)nitrosamine.

Hepatocyte sensitivity to initiation of carcinogenesis was studied as a function of the cell cycle phase in which damage was incurred. Hepatocytes were stimulated to proliferate by a two-thirds partial hepatic resection, and their proliferation was synchronized further by postsurgical treatment with hydrocortisone. Groups of male F344 rats were given a single administration of methyl(acetoxymethyl)nitrosamine, a highly reactive methylating agent, at various times after two-thirds partial hepatic resection when hepatocytes were in defined phases of the cell cycle. Beginning 3 wk after the treatment and for 37 wk thereafter, rats were fed a diet containing 0.05% phenobarbital to promote the expression of initiated hepatocytes. At 45 wk after treatment with carcinogen hepatocytic neoplasms were enumerated. The greatest yield of neoplasms (5.4 per liver) was observed in the group treated 16 h after two-thirds partial hepatic resection or at the time when proliferating hepatocytes began to enter the S phase of the cell cycle. The least yield of neoplasms (0.8 per liver) was identified in the group treated with methyl(acetoxymethyl)nitrosamine when hepatocytes were early in G1. In the proliferating hepatocytes sensitivity rose continuously during G1 to a peak at the G1-S border and then fell continuously as hepatocytes traversed S, G2, and M. This pattern of response could not be attributed to variation in hepatic esterase which activates methyl(acetoxymethyl)nitrosamine or to variation in methylation of DNA. The results support a model in which carcinogen-induced genetic alterations, occurring at the time of or soon after damaged cells enter the S phase, represent irreversible events that contribute to the initiation of carcinogenesis.

Animals↗