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Biomedical subjects

Z Darzynkiewicz

Publications and source records attributed to Z Darzynkiewicz.

At least 127 records · Page 7Linked to original sources
PubMed1995

Application of biotin, digoxigenin or fluorescein conjugated deoxynucleotides to label DNA strand breaks for analysis of cell proliferation and apoptosis using flow cytometry.

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deoxynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluoresceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.

Apoptosis↗
PubMed1995

Flow cytometric characterization of proliferation-associated nuclear antigen (p105) during the cell cycle in normal lymphocytes and promyelocytic leukemia cells (HL-60).

Bivariate flow cytometric analysis of p105 expression and DNA content was performed in human lymphocytes and promyelocytic leukemia cells (HL-60). We also employed a new method of detecting DNA strand breaks associated with apoptosis by labeling the 3'-OH termini in the breaks with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase (TdT). Phytohemagglutin-stimulated proliferating lymphocytes express p105 maximally after 48 hours, similar to HL-60 cells in exponential growth phase. Antigen expression in G1 was notably heterogeneous in G1 phase of both cell types and highest in M-phase cells treated for 6 hours with vinblastine. However, the p105-DNA ratio changed very little. Cycloheximide did not affect P105 expression. Methotrexate decreased p105 expression. Camptothecin and teniposide induced apoptosis, but apoptotic cells still expressed p105. Dual-parameter measurement also demonstrated that TdT-positive apoptotic cells expressed p105 at a higher level than apoptotic TdT negative cells. The data on drug treatment suggest that expression of p105 may be useful in monitoring chemotherapeutic effects but not as a marker of cell death from apoptosis.

Camptothecin↗
PubMed1995

Expression of cyclins A, D2 and D3 in individual normal mitogen stimulated lymphocytes and in MOLT-4 leukemic cells analyzed by multiparameter flow cytometry.

Cyclins are regulatory subunits of the cyclin dependent kinases (CDKs), the enzymes that drive the cell through the respective phases and check-points of the cell cycle. The expression of cyclins in non-tumor cells, regulated by timely induction of their synthesis and proteolysis, is scheduled, occurring at discrete periods of the cell cycle. Using multiparameter flow cytometry we have recently observed that expression of cyclins B1 and E in individual normal lymphocytes mitogenically stimulated by phytohemagglutinin (PHA) and lymphocytic leukemic MOLT-4 cells was similar, restricted to particular phases of the cycle: cyclin B1 was detected only in G2+M- and cyclin E in late G1 and early S-phase cells. In the present study we have measured the expression of cyclins A, D2 and D3 in these cells. The presence of cyclin A was restricted to late S and G2 phases, both in the case of lymphocytes and of MOLT-4 cells. Over 95% of the non-stimulated lymphocytes were both cyclin D2 and D3 negative. Mitogenic stimulation with PHA-induced expression of cyclins D2 and D3 in over 50% cells, which corresponds to the percentage of cells that respond to this mitogen in cultures. Expression of these proteins peaked between 8 and 24 h after addition of PHA, and then decreased at the time of cell entrance to S. During exponential growth (48-72 h after stimulation with PHA) expression of the D-type cyclins was diminished: only between 5-10% of the lymphocytes had levels of cyclin D3 as high as G1 cells between 8-24 h after PHA stimulation. Populations of proliferating lymphocytes and MOLT-4 cells were very heterogeneous in terms of expression of D-type cyclins by individual cells. While expression of cyclin D2 in exponentially growing MOLT-4 cells was similar to that of proliferating lymphocytes, the percent of cells expressing cyclin D3 as well as the degree of expression, was higher in MOLT-4 cells, regardless of the phase of the cycle. These results, with our earlier observations of the untimely expression of cyclins B1 and E in several other tumor lines, suggest that altered expression of cyclins may be a frequent feature of malignancy.

Analysis of Variance↗
PubMed1995

Induction of cell differentiation potentiates apoptosis triggered by prior exposure to DNA-damaging drugs.

At the end of their life span, differentiated cells die by apoptosis. Subsets of cells also die, in some cell systems, shortly after exposure to differentiating agents. This suggests that early during differentiation the cells may undergo "priming," during which synthesis and/or activation and accumulation of effectors of apoptosis occurs. The objective of the present study was to test the hypothesis that the signal for apoptosis provided by DNA-damaging drugs given prior to induction of differentiation will be more effective in triggering apoptosis than when given following induction of differentiation. Human promyelocytic HL-60 cells were treated with the topoisomerase I inhibitor camptothecin, the alkylating agent nitrogen mustard, or 5'-azacytidine, an antimetabolite affecting predominantly RNA metabolism. Following drug removal, the cells were postincubated with n-butyrate, which induces differentiation of HL-60 cells along the monocytic pathway, or with all-trans-retinoic acid, which triggers myelocytic differentiation. Multiparameter flow cytometry using two different methods of analysis of apoptosis-associated DNA breakage in situ, as well as evaluation of cell morphology and DNA gel electrophoresis, were used to ascertain the mode of cell death. Increases of 100-200% in the percentage of apoptotic cells were seen when cells were first treated with camptothecin or nitrogen mustard, followed by n-butyrate or retinoic acid, compared to the combined percentage of apoptotic cells when these agents were used individually.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimetabolites, Antineoplastic↗
PubMed1995

Growth imbalance and altered expression of cyclins B1, A, E, and D3 in MOLT-4 cells synchronized in the cell cycle by inhibitors of DNA replication.

Expression of cyclins at the translational level is generally studied by immunoblotting lysates of cells synchronized in the cycle. Most methods used to synchronize transformed cells induce growth imbalance. The aim of the present study was to analyze levels of cyclins B1, A, E, and D3 in the respective phases of the cycle in synchronized human leukemic MOLT-4 cells, correlate them with total cellular protein content (reflecting growth imbalance), and compare the synchronized cells with cells from unperturbed, asynchronous cultures. Expression of cyclins detected immunocytochemically in individual permeabilized cells was analyzed by multiparameter flow cytometry, which made it possible to relate position of the cell in the cell cycle with cyclin expression. Cells synchronized at the G1-S boundary by thymidine, mimosine, or aphidicolin had about 40% increased total protein and 4-5 fold higher levels of cyclins E and B1 compared to their G1 counterparts from unperturbed cultures. Expression of cyclin A in synchronized cells was 2-fold higher, while expression of cyclin D3 was essentially unaltered. The synchronized cells traversing S phase after release from the block had elevated but decreasing levels of cyclins E, B1, and A. Although the cyclin expression of cells reentering G1 was similar to that of their counterparts from asynchronous cultures, the total protein content was still elevated by about 30%. The data indicate that due to different degrees of imbalance in total protein and individual cyclin content, levels of cyclins detected by immunoblotting of cell lysates from synchronized cultures may not be representative of their expression in unperturbed cells. The elevated level of cyclin B1 in the cells arrested at the G1-S boundary may reflect the increased half-life of this protein, stabilized as the result of the overexpression of cyclin E.

Cell Cycle↗
PubMed1995

Induction of apoptosis and cell cycle-specific change in expression of p53 in normal lymphocytes and MOLT-4 leukemic cells by nitrogen mustard.

DNA damage in the cell activates expression of the p53 tumor suppressor gene, whose role is associated with cell arrest in G1 or apoptosis. The aim of this study was to examine the cell cycle position-related changes in expression of p53, as well as induction of apoptosis, in mitogen-stimulated normal human lymphocytes and in human leukemic MOLT-4 cells (which express mutated p53), following DNA damage by the alkylating agent nitrogen mustard. Measurement of p53 expression and DNA content by flow cytometry followed by bivariate analysis of the data made it possible to correlate the drug-induced changes in p53 expression in individual cells with their cell cycle position without the need for cell synchronization. Expression of p53 was detected immunocytochemically using the AB-6 mAb, which reacts with the product of the wild-type p53 tumor suppressor gene and with most of its mutated forms. Exposure of normal lymphocytes to 5 microM nitrogen mustard caused their arrest in G1, an increase in p53 expression which was maximal in such cells, and significant apoptosis in cells located beyond the arrest point (S and G2 + M cells). In contrast, neither arrest in G1 nor significant apoptosis of MOLT-4 cells was seen after administration of either 0.5 or 5 microM nitrogen mustard for up to 24 h, although the drug reduced the rate of cell progression in the S-phase at both concentrations. Expression of p53 was highest for S and G2 + M MOLT-4 cells in response to the nitrogen mustard. Although a severalfold lower level of p53 was detected in lymphocytes compared to MOLT-4 cells prior to drug treatment, the relative increase in p53 expression in response to the drug was 2-fold higher in lymphocytes. These data suggest that DNA damage caused by nitrogen mustard provides a signal that results in stabilization of wild-type p53, preferentially in G1 cells, causes cell arrest in G1, and induces apoptosis of the cells that either were in the S-phase at the time of drug administration and/or escaped G1 arrest. The increase in expression of mutated p53, in response to DNA damage, is unrelated to the cell cycle position, and neither provides a signal for cell arrest in G1 nor a trigger for immediate apoptosis.

Apoptosis↗
PubMed1994

Unscheduled expression of cyclin B1 and cyclin E in several leukemic and solid tumor cell lines.

Normal, nontumorous cells express cyclin proteins in an orderly, scheduled fashion, at a given phase of the cell cycle. Thus, cyclin B1 is synthesized during G2 and abruptly degraded during mitosis. The onset of cyclin E synthesis takes place in mid-G1, its maximal expression is at the time of cell entrance to S, and its degradation occurs during cell progression through S phase. In the present study, multiparameter flow cytometry was used to correlate expression of cyclin B1 or cyclin E with cell cycle position (estimated by cellular DNA content) in normal human proliferating lymphocytes as well as in T-cell MOLT-4 leukemia; promyelocytic HL-60 leukemia; histiocytic U937 lymphoma; MCF-7, T-47D, and Hs 587T breast carcinoma; Colo 320DM colon carcinoma; and the T-24 transitional cell carcinoma cell line. The scheduled expression of both cyclins, namely of cyclin B1 restricted to G2 + M cells and of cyclin E restricted to late G1 and early S cells, was observed only in normal lymphocytes and MOLT-4 cells. The cells of HL-60, U937, T-47D, and Hs 587T lines expressed both cyclins in an unscheduled ("ectopic") fashion, i.e., unrelated to cell cycle position. Colo 320DM cells showed unscheduled expression of cyclin E (i.e., during G2) but expression of cyclin B1 in this line was generally restricted to G2 + M cells. There were relatively few (10-12%) cells in MCF-7 and T-24 cell lines that expressed cyclin B1 or E in an unscheduled manner. It may be expected that the unscheduled expression of cyclins in tumor cells may lead to a loss of the regulatory mechanisms of cell cycle progression and that such feature of the tumor may be of prognostic value. There is a need, therefore, to conduct similar studies in primary tumor cells.

Cell Cycle↗
PubMed1994

Simultaneous analysis of DNA replication and apoptosis during treatment of HL-60 cells with camptothecin and hyperthermia and mitogen stimulation of human lymphocytes.

A new method is described which combines the identification of DNA replicating and apoptotic cells in a single measurement by flow cytometry. The detection of DNA replicating cells is based on incorporation of 5-bromo-2'-deoxyuridine or 5-iodo-2'-deoxyuridine, followed by selective photolysis at the site of incorporation of the halogenated DNA precursors. Single-strand breaks in DNA resulting from the photolysis are subsequently labeled with digoxygenin or biotin-conjugated dUTP in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The double-stranded DNA breaks in apoptotic cells resulting from activation of the endonuclease can be labeled in this reaction as well. However, in contrast to the photolysed DNA, the low molecular weight fraction of DNA of apoptotic cells is extractable from the cells, and the degree of DNA elution can be modulated by cross-linking with formaldehyde. Thus, apoptotic cells can be distinguished and quantified by virtue of their fractional DNA content. Replication of less than 1% of a genome of a cell in the presence of 5-bromo-2'-deoxyuridine (equivalent of a 5-min 10 microM 5-bromo-2'-deoxyuridine pulse) can be detected by the selective photolysis method. The method was applied to study apoptosis and proliferation of human leukemic HL-60 cells and normal, mitogen-stimulated lymphocytes. Whereas apoptosis of HL-60 cells induced by the DNA topoisomerase I inhibitor camptothecin was selective to DNA replicating cells, apoptosis induced by hyperthermia showed no such selectivity. Lymphocytes that preferentially underwent apoptosis in cultures stimulated by phytohemagglutinin did not initiate DNA replication. By offering the possibility for identification of both DNA replicating and apoptotic cells in a single measurement, the method may find an application in studies of the prognostic value of both cell proliferation and death in human tumors and the apoptotic response of DNA replicating vs. nonreplicating cells to different treatments.

Apoptosis↗
PubMed1994

Staurosporine blocks cell progression through G1 between the cyclin D and cyclin E restriction points.

The protein kinase inhibitor staurosporine (SSP) stops progression of normal nontransformed cells in the G1 phase of the cell cycle. This implies that at least one of the cell cycle associated kinases, essential for cell transit through G1, is sensitive to SSP. Using multivariate flow cytometry to correlate the expression of cyclin E or cyclin D with cellular DNA content (i.e., cell cycle position), we have presently characterized the point of action of SSP in relation to the expression of these cyclins. During stimulation of normal human lymphocytes by phytohemagglutinin, cyclin D was expressed early, peaking at 8-14 h, while cyclin E appeared later, reaching a maximum at the time of cell entrance to S phase (24 h). Addition of SSP at the time of cell stimulation, while markedly suppressing the expression of cyclin E, had a rather modest effect on the expression of cyclin D. The data indicate that the SSP sensitive kinase(s) involved in cell progression through G1 operate beyond the restriction point of cyclin D but prior to that of cyclin E. Thus, the target(s) of SSP is (are) either the p33cdk/cyclin E complex itself or other protein kinase(s), activated subsequent to the cyclin D but prior to the cyclin E restriction point, the activity of which is essential for cell transit through G1.

Alkaloids↗
PubMed1994

A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry.

In cells undergoing apoptosis (programmed cell death), a fraction of nuclear DNA is fragmented to the size equivalent of DNA in mono- or oligonucleosomes. When such DNA is analyzed by agarose gel electrophoresis it generates the characteristic "ladder" pattern of discontinuous DNA fragments. Such a pattern of DNA degradation generally serves as a marker of the apoptotic mode of cell death. We developed a simple, rapid, and selective procedure for extraction of the degraded, low-molecular-weight DNA from apoptotic cells. The cells are prefixed in 70% ethanol, DNA is extracted with 0.2 M phosphate-citrate buffer at pH 7.8, and the extract is sequentially treated with RNase A and proteinase K and then subjected to electrophoresis. The ladder pattern was detected from DNA extracted from 1-2 x 10(6) HL-60 cells, of which as few as 8% were apoptotic, by flow cytometric criteria, as well as from blood and bone marrow samples from leukemic patients undergoing chemotherapy. The method is rapid and uses nontoxic reagents (no phenol, chloroform, etc.). This approach permits the analysis of DNA extracted from the very same cell population that is subjected to measurements by flow cytometry to estimate DNA ploidy, the cell cycle distribution of nonapoptotic cells, the percentage of apoptotic cells, or other parameters. Furthermore, the cells may be stored in 70% ethanol for at least several weeks before analysis without any significant DNA degradation. Treatment with ethanol also inactivates several pathogens, thereby increasing the safety of sample handling. The method is applicable to clinical samples, which can be fixed in ethanol and then stored and/or safety transported prior to analysis.

Apoptosis↗
PubMed1994

Flow cytometric detection of apoptosis: comparison of the assays of in situ DNA degradation and chromatin changes.

The aim of this study was to compare three methods of detection of apoptotic cells: (1) the method based on elution of low molecular weight DNA from the ethanol fixed cells followed by cell staining with DAPI (diamidino-2-phenylindole) or propidium iodide as the DNA fluorochromes, (2) the method of in situ labeling of DNA strand breaks with biotinylated dUTP, utilizing exogenous terminal deoxyribonucleotide transferase, and (3) the method of analysis of DNA denaturation in situ using acridine orange to differentially stain denatured and double-stranded DNA sections following cell exposure to 0.1 M HCl. Cells of the human promyelocytic HL-60 line, treated in vitro with the DNA topoisomerase I inhibitor camptothecin, which selectively triggers apoptosis of S-phase cells, were chosen as a model. The method based on analysis of changes in DNA denaturability was the most sensitive in terms of detection of the earliest changes in chromatin of cells undergoing apoptosis; the increased sensitivity of DNA to denaturation in S-phase cells was measured as early as 100 min after addition of camptothecin. DNA cleavage, assayed either by the univariate measurement of DNA content following extraction of low molecular weight DNA, or by labeling DNA strand breaks with biotinylated dUTP, was detected in S-phase cells after 120 min incubation with camptothecin. The percentage of apoptotic cells at the late stage of apoptosis, the kinetics of cell transition to apoptosis, and kinetics of the loss of S phase cells were all essentially similar when measured by any method.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis↗
PubMed1994

Detection of apoptosis-associated DNA strand breaks in fine-needle aspiration biopsies by in situ end labeling of fragmented DNA.

The predominant mode of either spontaneous or drug-induced death of cells in tumors is apoptosis. A flow cytometric method was developed in our laboratory to identify apoptotic cells, based on labeling DNA strand breaks, which appear as a result of extensive DNA cleavage by the apoptosis-associated endonuclease, with biotinylated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The aim of this study was to reveal whether this methodology can be applied to human solid tumors sampled by fine-needle biopsy. Twenty-two tumors, consisting of 11 breast carcinomas; three metastatic anaplastic carcinomas; three adenocarcinomas of colon, endometrium, and lung; two metastatic lymph node squamous cell carcinomas of the larynx; and three malignant lymphomas were examined. It was possible to identify cells with DNA strand breaks in all these tumors. Extremely high variability in the proportion of cells with DNA strand breaks was observed between the individual tumors. In diploid tumors (n = 12) the percentage of cells with DNA strand breaks varied from 1% to 43%, and the mean value was 19%. In aneuploid tumors this percentage varied from 15% to 51% and the mean value was 37%. In the latter tumors the presence of cells with DNA strand breaks was limited to the DNA aneuploid cell population; very few diploid, presumably tumor infiltrating or stromal cells, showed the presence of DNA strand breaks. No correlation was observed between the percent of cells in S phase and those with DNA strand breaks. The data indicate that apoptosis is more frequent in populations of tumor cells than among normal cells of the same organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1994

CD4 engagement induces Fas antigen-dependent apoptosis of T cells in vivo.

CD4 is a T lymphocyte receptor for major histocompatibility complex class II antigens. It is referred to as coreceptor because it synergizes with the T cell receptor for antigen when both receptors become engaged simultaneously. We show here in mice that when engaged by antibody independently of the T cell antigen receptor, CD4 induces T cells to undergo apoptosis. Several features of this process were identified. The expression of an intact Fas protein is a requirement for CD4-mediated T cell death. Mice homozygous for the lpr mutation which are defective in the expression of Fas and in their ability to delete lymphocytes apoptotically fail to delete anti-CD4-reactive T cells. Sessile anti-CD4-reactive T cells leave their homing environment in lymphoid organs and modulate their cell surface molecules, e.g. CD2, CD3, CD4. A massive influx of lymphoid cells with null-cell phenotype occurs in the blood where they begin to reexpress cell surface markers. With their arrival in the circulation, anti-CD4-reactive T cells develop features of DNA degradation typical of apoptosis. More than one third of the circulating lymphoid cells show apoptotic features 7-8 h after anti-CD4 injection. Their frequency declines subsequently presumably due to their physical disintegration via shedding of apoptotic bodies and phagocytosis. Our data show that when not obliged to the activation process by the antigen receptor, CD4 can mediate deletion signals. Thus, besides functioning as coreceptor with the antigen receptor, CD4 has a function of its own in facilitating the induction of apoptosis.

Animals↗
PubMed1994

Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis.

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G1, most virus transformed or tumor cells are unaffected in G1 but arrest in G2 phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nuclear p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 microM SSP. MOLT-4 cells did not arrest in G1 or G2 phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G1 phase at higher DNA ploidy (tetraploidy; G1T), and then progressed through ST (rereplication) into G2T and MT. The rates of entrance to G2 and G2T were essentially identical, indicating that the rates of cell progression through S and ST as well as through G2 and G2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 microM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis.

Alkaloids↗
PubMed1994

Apoptosis in adult retinal ganglion cells after axotomy.

Lesions to the mature mammalian central nervous system cause irreversible degeneration, in which neurons have been previously thought to be passive victims. In this study, axon-lesioned adult rat neurons are shown instead to actively degrade themselves through the process of apoptosis: a programmed type of cell death in which the cellular apparatus is actively involved in the degradation process. To investigate whether retinal ganglion cells of an adult mammal follow an apoptotic type of death when their axons are severed, DNA breaks in nuclei were labeled in situ, using a method that specifically incorporates biotinylated deoxynucleotides by exogenous terminal deoxynucleotidyl transferase on the 3'-OH ends of DNA. The active nature of the death mechanism was demonstrated by the reduction in biotin-labeled nuclei after administering the protein synthesis inhibitor cycloheximide. Our results suggest that retinal ganglion cells of the adult rat die through apoptosis when axotomized. This raises new possibilities in the treatment of CNS injuries, by the potential interruptibility of a program for neuronal death.

Animals↗
PubMed1994

Presence of neuronal proteins in serially cultured cells from autopsy human brain.

Cells that contain the high (NF-H) and medium (NF-M) molecular weight neurofilaments and neuron-specific enolase (NSE) have been cultured from human brain (Brodmann frontal area 9 and hippocampus) obtained at routine autopsy. Three of these cultures (from a 52-year-old man with vascular dementia, a 75-year-old man with Alzheimer's disease, and an 80-year-old normal woman) have been propagated to passage 12-14 and studied in detail. Cellular morphology was pleomorphic and consistent with a relatively low level of differentiation, by both light microscopic and ultrastructural examination. Immunocytochemical reactivity in a characteristic filamentous pattern was observed with monoclonal antibodies to nonphosphorylated epitopes of NF-H and NF-M as well as with redundant antibody probes specific for NF-H and NF-M. Immunoblotting studies confirmed the presence of NF-H and NF-M. Immunocytochemistry and Immunoblotting also demonstrated the presence of NSE, but not of glial fibrillary acidic protein or other glial markers. Immunocytochemical studies also indicated the presence of proline hydroxylase, an enzyme found in fibroblasts but not neurons. These studies indicate that cells can be cultured from autopsy human brain which are not neurons but nevertheless express molecules used as markers (NF-H, NF-M, and NSE) of adult neurons in vivo and in culture.

Aged↗