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R E Cunningham

Publications and source records attributed to R E Cunningham.

34 records · Page 2Linked to original sources
PubMed1993

bcl-2 rearrangement in Hodgkin's disease. Results of polymerase chain reaction, flow cytometry, and sequencing on formalin-fixed, paraffin-embedded tissue.

We examined 81 cases of Hodgkin's disease for evidence of the t(14;18) translocation, using the polymerase chain reaction assay on lysates of formalin-fixed, paraffin-embedded tissue. Seven of 74 amplifiable cases (9%) were positive for the translocation, which involves the bcl-2 oncogene and the immunoglobulin heavy chain gene. Two of these cases were sequenced and the breakpoints had the same pattern found in follicular lymphoma. The nuclei from one of the cases were sorted into large and small subpopulations. The t(14;18) signal was more intense in the large nucleus subpopulation, which contained a greater proportion of Reed-Sternberg-like nuclei. These results are consistent with the hypothesis that Reed-Sternberg cells carry the translocation, but they do not exclude the possibility that the translocation is found in cells representing the reactive component of Hodgkin's disease. The results also demonstrate that routinely processed material is suitable for polymerase chain reaction-based analysis of translocations, although the sensitivity is reduced 10- to 100-fold, compared with fresh tissue.

Base Sequence↗
PubMed1989

Prognostic significance of DNA ploidy in carcinoma of prostate.

Flow cytometry was used to measure the DNA content in archived paraffin-embedded human prostatic cancer tissue for 69 patients with known outcomes that presented between 1975 and 1982. Of these, 51 patients had clinically localized lesions and were surgically staged prior to radical prostatectomy, while 18 patients presented with advanced Stage D2 disease. Thirty-six of 37 (97.3%) pathologic Stage B lesions were diploid. In contrast, the majority (72.2%) of patients with metastatic disease had aneuploid tumors. The average Gleason grade for aneuploid tumors was 8.2 +/- 1.98 versus 5.5 +/- 1.89 for diploid tumors (p less than 0.01). For 51 patients with clinically localized tumors, 13.9 percent of diploid tumors with a low Gleason sum (2 to 6) had extracapsular spread of tumor or regional lymph node involvement compared with 83.3 percent of aneuploid tumors with high Gleason scores (7 to 10). The addition of DNA ploidy to degree of glandular differentiation may enhance the prognostic evaluation of prostatic tumors and eventually improve our ability to select patients who are likely to benefit from radical prostatectomy.

Aged↗
PubMed1988

Expression of genes for platelet-derived growth factor in adult human venous endothelium. A possible non-platelet-dependent cause of intimal hyperplasia in vein grafts and perianastomotic areas of vascular prostheses.

Neointimal fibromuscular hyperplasia (NFH) in vein grafts and perianastomotic zones of vascular prostheses has been attributed to the effects of platelet-derived growth factor (PDGF) released by platelets interacting with bypass conduits. But inhibition of platelet aggregation often fails to prevent NFH, and recurrent growth of intact, platelet-free endothelium over perianastomotic areas where NFH occurs is inconsistent with the concept of sustained PDGF release from platelets causing NFH progression at late times after surgical procedures. Cultured bovine aortic endothelial cells (ECs) and human umbilical vein ECs have been shown to release a PDGF-like molecule. We report that confluent cultured fourth passage adult human saphenous vein ECs (AHSVECs) grown in the presence of heparin (100 micrograms/ml) and retina-derived growth factor (RDGF) studied by Northern blotting transcribed a messenger ribonucleic acid (mRNA) of 3.9 kb, strongly hybridizing to PDGF B chain probes, and two species of 2.0 and 2.6 kb hybridizing to PDGF A chain probes. Withdrawal of RDGF and heparin from these cultures for 48 hours before mRNA extraction amplified the scanning densitometric mRNA signal per cell by 8.0 +/- 7.6 fold (mean +/- SD) (N = 4 cultures) for B chain mRNA and 5.2 +/- 3.6 fold (N = 3 cultures) for A chain mRNA. In addition, AHSVEC cultures released a PDGF-like substance, because 50% vol/vol AHSVEC-conditioned serum-free medium increased tritiated thymidine uptake elevenfold in PDGF receptor-bearing 3T3 cells whereas an excess (50 micrograms/ml) of nonspecific goat anti-human-PDGF antibody significantly reduced this increase by a mean of 30% to 7.0 +/- 3.4 fold (N = 6 trials, p less than 0.001). Flow cytometry determined AHSVEC cultures to be proliferating with a mean of 6.2% +/- 1.9% (N = 3 culture lines) of ECs in S phase even at confluence when deprived of EC mitogens for 48 hours. Adult human ECs, which proliferate on bypass conduits and host vessels after perioperative injury, may play a role in causing NFH by stimulating proliferation of adjacent smooth muscle cells. Prevention of NFH may require not only antiplatelet agents but also ways to prevent EC release of smooth muscle cell mitogens in response to perioperative EC injury.

Animals↗
PubMed1987

Cell growth cycle block of T cell hybridomas upon activation with antigen.

Stimulation of antigen-specific T cell hybridomas with the appropriate antigen/MHC combination, at concentrations that resulted in the secretion of the lymphokine interleukin 2, resulted in a dose-dependent decrease in both [3H]thymidine incorporation and cell growth. Flow cytometric studies demonstrated that stimulation with antigen resulted in a cell cycle block that was most evident at the G1/S border, and mixing studies revealed that bystander T cells of different antigen specificities were not affected. For at least the large majority of T cells, the G1/S cell cycle block appeared to be irreversible after 24 h of exposure to antigen. This cell cycle block may be useful as a rapid and quantitative measure of T cell hybridoma activation, as a means of selecting T cell hybridomas that have functional alterations in the reception of stimulatory signals, and may serve as a model of the induction of tolerance in immature T cells.

Animals↗
PubMed1987

Heterogeneous expression of a murine B16 melanoma-associated antigen correlates with cell cycle.

A murine monoclonal antibody (MM2-3C6) that reacts with a B16 murine melanoma-associated membrane antigen was used to study the relationship of antigen expression to the cell cycle. Dual-parameter flow cytometric measurements of membrane antigen and DNA revealed that antigen-positive cells were present throughout the cell cycle. Peak antigenic expression was noted during the late log phase of the cell growth curve with negligible antigen-negative population. The emergence of a distinct antigen-negative population (30%-40%) was noted in the late stationary phase. Cell cycle analysis indicated that the negative subpopulation was restricted to the G0/G1 phase, thus, demonstrating antigenic heterogeneity within the tumor cell population. Cell sorting was performed to analyze the origin of such heterogeneity. Following two sequential sortings, the antigen-negative cells became antigenic upon reculture. Again, at the late stationary phase, a distinct antigen-negative population (30%-40%) emerged. The sorted antigen-positive cells showed flow cytometric profiles identical to the sorted antigen-negative population upon reculture. Therefore, in this murine model, it appears that antigen expression is cell cycle dependent and such expression seems to be associated with proliferation.

Animals↗
PubMed1985

The use of flow cytometry and cell sorting in a human colon carcinoma model.

We investigated the growth characteristics of a human colon carcinoma cell line, WiDr, grown in culture flasks and on chick embryonic skin (CES). WiDr cells labeled in vitro with bromodeoxyuridine (BrdU) and analyzed by combined propidium iodide/Hoechst 33258 fluorescence showed evidence of more BrdU incorporation in early S phase as compared to late S phase. When inoculated on the CES, WiDr cells multiplied and invaded the underlying skin. Morphologic examination showed that with extended culture WiDr cells on the CES undergo progressive structural organization with the development of acini and basal lamina, structures similar to those in in vivo tumors. WiDr cells were labeled with monoclonal antibody to carcinoembryonic antigen (CEA) and the brightest 2% of the population was sorted. When subsequently grown on the CES, the sorted cells formed significantly more acinar structures at 3 and 6 days of culture than an unsorted population grown for a comparable time.

Animals↗
PubMed1985

Expression of a murine B16 melanoma-associated antigen analyzed by flow cytometry.

Understanding factors regulating expression of tumor antigens is vital to the design of rational immunotherapeutic strategies. A murine monoclonal antibody (MM2-3C6) that recognizes a B16 murine melanoma-associated membrane antigen was used to study antigen expression in relation to cell cycle, clonal heterogeneity, and growth density. Dual-parameter flow cytometric measurements of DNA content and membrane antigen demonstrated that antigen-positive cells were found throughout the cell cycle. However, some antigen-negative cells were observed and were restricted to the G0/G1 phase demonstrating the antigenic heterogeneity of this tumor line. Three sublines of B16, F1, F10, and F1r with variable metastatic potential, and 50 B16 F1 clones all expressed the antigen with a mean antigen density above background of 8.84 +/- 2.52 [( mean cell fluorescence/cell size] X 10(-3] ranging from 3.68 to 16.57. Further studies of three sublines and five clones showed antigen density fluctuated over an 8-day growth period in culture with daily changes of medium. During log growth from Day 1 (1.2 +/- 0.08 X 10(4) cells/cm2) to Day 4 (20.4 +/- 3.01 X 10(4) cells/cm2), antigen density increased from 4.09 +/- 0.29 to 6.86 +/- 0.29. By Day 8 (26.5 +/- 2.86 X 10(4) cells/cm2) when the cells had been confluent for 3 days, antigen density decreased to 2.54 +/- 0.26 which was significantly lower than other days measured (P less than 0.05). It is concluded that tumor cell proliferation and cell density can modulate antigen expression in this system and, therefore, this may be a useful model to study tumor cell escape from immunotherapy.

Animals↗
PubMed1984

Colony-stimulating factor (CSF) controls proliferation of CSF-dependent cells by acting during the G1 phase of the cell cycle.

Proliferation of granulocyte/macrophage (GM) progenitor cells in soft agar cultures is dependent on the continuous presence of colony-stimulating factors (CSF). To elucidate this dependency we studied the effect of deprivation and readdition of CSF on the cell cycle kinetics of a GM-CSF-dependent murine mast/basophil cell line (PT-18). Flow cytometry and [3H]thymidine incorporation have been used to analyze the complete cell cycle. Removal of CSF from the culture medium resulted in accumulation of the cells in the G1 phase. Eighteen hours after removal of CSF, 85% of the cells were arrested in G1 phase. Readdition of GM-CSF to such quiescent cells was followed by progression of the cells from G1 into S phase with a lag period of 10 hr. A similar lag period was observed when cells released from G2+M arrest progressed, in the presence of CSF, to S phase via the G1 phase. These findings indicate that deprivation of GM-CSF does not move PT-18 cells out of the cycle to a G0 phase but rather arrests them at early G1 phase. Finally, we demonstrate that, for the cells to progress through the cell cycle, the requirement for the presence of GM-CSF is limited to the first 6 hr of the 10-hr duration of the G1 phase.

Animals↗
PubMed1984

Regulation of interleukin 2 receptor expression: effects of phorbol diester, phospholipase C, and reexposure to lectin or antigen.

Anti-Tac monoclonal antibody identifies the receptor for interleukin 2 (IL 2, or T cell growth factor) present on activated human T lymphocytes. By using tritiated anti-Tac, we now report a sensitive and specific binding assay to evaluate cell surface IL 2 receptor expression. IL 2 receptors on human peripheral blood lymphocytes can be detected within 6 hr after PHA stimulation. PHA-induced receptor expression is inhibited by actinomycin D and cycloheximide, but not by mitomycin C, suggesting a requirement for de novo RNA and protein synthesis, but not DNA synthesis. Scatchard analysis of [3H]-anti-Tac binding to lymphocytes stimulated with PHA for 3 days revealed from 20,000 to 60,000 molecules of antibody bound per cell, and a Kd of 1 to 3 x 10(-10) mol/l. Sequential binding studies of activated human lymphocytes maintained in long-term culture with IL 2 demonstrated a progressive decline in receptor number correlating with diminished growth rate. Restimulation with lectin or antigen increased the number of IL 2 receptors, suggesting that IL 2 dependent immune responses may be regulated, at least in part, by IL 2 receptor expression. Receptor number was also increased by PMA. Moreover, similar effects were produced by incubation with phospholipase C but not interleukin 1. Because both PMA and phospholipase C result in activation of protein kinase C, these data suggest the possibility that activation of protein kinase C may induce IL 2 receptor expression.

Antigens, Surface↗
PubMed1982

Artifacts associated with mithramycin fluorescence in the clinical detection and quantitation of aneuploidy by flow cytometry.

Ethanol-fixed cells stored at 4 degrees C exhibit fixation time-dependent hyperchromatism in comparison with freshly fixed cells when stained with mithramycin and examined by flow cytometry. This hyperchromatism has been found to be temperature-dependent, developing fully within 72 hr at room temperature, and within 2 hr at 37 degrees C. Cells from normal donors that are stained with mithramycin exhibit spurious aneuploid peaks. These spurious aneuploid peaks can be eliminated by incubating ethanol-fixed cells at 37 degrees C for 2 hr prior to staining; true aneuploidy is not affected by this procedure. In rare instances, cytoplasmic fluorescence can be observed in mithramycin-stained cells. In addition, unexplained hypochromatism and hyperchromatism can be observed in some clinical samples, particularly in human melanoma. The effects of these unexplained staining artifacts can be minimized or eliminated by adopting strict criteria for the clinical detection of aneuploidy by flow cytometry.

Aneuploidy↗
PubMed1981

Kinetic effects of sangivamycin in sarcoma 180 in vitro.

The lethal and sublethal effects of sangivamycin (SGM) were studied in sarcoma 180 in vitro in relation to drug concentration and duration of drug exposure. SGM lethality was found to be dependent on both drug concentration and duration of drug exposure. Pronounced effects on cell survival were observed only when SGM exposure was prolonged; with prolonged drug exposure, small increments in SGM concentration resulted in large increases in cell killing. Log-phase cells were more susceptible to the lethal effects of SGM than were early-plateau-phase cells. Measurements of incorporation of [3H]thymidine and [3H]uridine into the acid-insoluble cell fraction demonstrated inhibition of both DNA and RNA synthesis by SGM which was also dependent on drug concentration and duration of drug exposure, reflecting the lethality characteristics of SGM. As SGM concentration was increased, DNA synthesis was inhibited more rapidly than was RNA synthesis. Flow cytometry demonstrated a concentration- and time-dependent accumulation of cells in the late S and G2-M region of the DNA histogram. Our findings indicate that maximum lethality is obtained by prolongation of SGM exposure, and they suggest that pharmacokinetic studies may be important for determining regimens which provide such exposure in humans.

Animals↗
PubMed1981

Schedule-dependent synergism of combinations of hydroxyurea with adriamycin and 1-beta-D-arabinofuranosylcytosine with adriamycin.

The lethal effects of combinations of adriamycin (ADR) and either hydroxyurea (HU) or 1-beta-d-arabinofuranosylcytosine (ara-C) were studied in relation to drug-scheduling interval in Sarcoma 180 cells grown in vitro. Drug lethality was determined by cell cloning in soft agar. Serial kinetic changes induced by a priming dose of HU or ara-C were monitored by flow cytometry and related to schedule-dependent cell killing by ADR. All drug exposures were for 1 hr. When ADR was given together with either HU or ara-C, cell log kill was additive. However, when ADR was given after exposure to either HU or ara-C, cell killing was increased up to 200- to 500-fold, respectively. Maximum schedule-dependent synergism was observed at a drug-scheduling interval of 2 hr; schedule-dependent synergism decreased as the interval between drugs was increased beyond 2 hr. Schedule-dependent synergism was not observed when the same drug combinations were given in reversed order. Drug-induced changes in the DNA histogram were not seen until 5 hr after HU exposure and 8 hr after ara-C exposure. Thus, the schedule-dependent synergism between ADR and either HU or ara-C cannot be explained by cell cycle blockade with synchronization of cells in S phase.

Animals↗
PubMed1980

Dual parameter flow cytometry studies in human lymphomas.

Dual parameter flow cytometry studies using Coulter volume and cell DNA content were carried out in monodisperse cell suspensions of 64 samples of human lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, and benign lymphoid proliferations. Differences in mean Coulter volume among the lymphomas were due both to the intrinsic differences in mean G1 cell Coulter volume and to the presence of increased fractions of larger S and G2 cells, especially among the large B cell lymphomas. However, the relative contribution of large non-G1 cells to the overall population Coulter volume distribution was a relatively minor one; the presence of cells in S did not increase mean Coulter volume by more than 10%, even in samples with high S fractions. There was a good correlation between mean G1 cell Coulter volume and the log of the fraction of cells in S among the B cell lymphomas (r = 0.55). Evidence is presented that within individual samples, large cells proliferate more rapidly than small cells. This was seen in every case, both in the normal samples and in the lymphomas, and in the T cell lymphomas as well as in the B cell lymphomas. Aneuploidy was detected by flow cytometry in 11 cases; in 7 cases the aneuploid cell component could be analyzed separately from the diploid cell component on the basis of cell Coulter volume differences. The aneuploid components of diploid-aneuploid mixtures had higher S fractions than the diploid components in six of seven cases (0.16 +/- 0.04 [SE] vs. 0.08 +/- 0.02). These findings are considered in relation to the histopathological classification of the lymphomas, and in relation to the concept of clonal selection and clonal evolution of tumors.

Aneuploidy↗