L. David Tomei

Epidermal Growth Factor and tumor promoters prevent DNA fragmentation by different mechanisms

Serum deprivation of C3H 10T 1/2 fibroblasts resulted in DNA fragmentation which was prevented by growth factors such as Epidermal Growth Factor or the tumor promoters, 12-0-tetradecanoyl-13-0-phorbol acetate and Dihydroteleocidin B. Palmityl carnitine, an inhibitor of Ca2+-phospholipid-dependent protein kinase C, reversed the effects of the tumor promoters, but not the effect of Epidermal Growth Factor.

Psychological stress and phorbol ester inhibition of radiation-induced apoptosis in human peripheral blood leukocytes

Apoptosis is a process of genetically programmed alterations of cell structure that lead to failure of proliferation and differentiation, and eventual cell death. Apoptosis is induced by a variety of toxic insults including growth factor deprivation and ionizing radiation. This process may function to protect against the appearance of heritable phenotypic changes in cells and may be a critical factor in normal cellular immune function. Phorbol esters inhibit apoptosis, but little is known about factors that regulate this process physiologically. In this study, we demonstrate an association between an acute psychological stressor, taking examinations, and the induction of substantial and reversible changes in the response of peripheral blood leukocytes to gamma irradiation and to phorbol ester treatment. These data suggest that psychological stress may induce physiological changes that regulate the ability of immune cells to initiate apoptosis.

Role of prostanoids and lipid peroxides as mediators of the 12-O-tetradecanoylphorbol-13-acetate effect on cell growth

Confluent cultures of guinea pig smooth muscle cells (SMC) or human fibroblasts (HNF) were treated with 12-tetradecanoylphorbol-13-acetate (TPA). Prostanoid levels were measured by the radioimmunoassay of 6-keto-PGF1α and PGE2, and lipid peroxides were measured by the thiobarbituric acid test for malondialdehyde (MDA). Cells were seeded at low densities, and growth was calculated both from the cell count (Coulter Counter) and the colony number (image analysis). When confluent SMC and HNF were incubated in media alone, 6-keto-PGF1α levels were a function of the TPA concentration, increasing to a maximum at 10−8 M TPA and then decreasing at higher TPC concentrations. When confluent SMC and HNF were incubated in media containing exogenous arachidonic acid, 6-keto-PGF1α levels again increased to a maximum at 10−8M TPA but decreased at higher TPA concentrations only with SMC. The increase in 6-keto-PGF1α levels was much greater in HNF (1310%) than SMC (680%). SMC synthesized similar amounts of 6-keto-PGF1α and PGE2, and the stimulatory effect of TPA was similar with 6-keto-PGF1α and PGE2. Indomethacin (IM) blocked prostanoid synthesis at all TPA concentrations. TPA did not have a significant effect on MDA levels in either cell line. The lipid antioxidants α-tocopherol and α-tocopherylquinone blocked lipid peroxidation without affecting the stimulation of prostanoid synthesis with TPA. Cell number decreased to a minimum at 10−8M TPA in both cell lines. The decrease in cell number was much greater in HNF (72%) than SMC (30%). SMC colony number decreased at 10−8 TPA and then increased at 10−6M TPA. IM did not block the TPA effect on cell number in either cell line. The lipid antioxidant butylated hydroxytoluene (BHT) did not block the TPA effect on SMC cell number. The IM and the BHT data show that the TPA effect on cell growth is not mediated by prostanoid or lipid peroxide products of arachidonic acid metabolism. However, the increase in prostanoid synthesis parallels the decrease in cell number, and the effects are maximal at the same TPA concentration. These correlations suggest a common cellular process affecting both prostanoid synthesis and cell growth that is initiated or enhanced by TPA.

Performance and applications of the dynamic focus scanning laser imaging system

A multipurpose digital imaging device has been developed for applications in quantitative densitometry, light scatter detection and fluorescence emission measurement. The Scanning Laser Imaging (SLI) device consists of a three-dimensional laser beam controller, a fiber optic faceplate coupler, a specially designed photodetector assembly and a support computer that has interactive control over the beam positioner. Image information is collected as measurement of either total forward light loss, forward direction scattering or fluorescent emission, depending on the nature of the biological target and the configuration of the detection assembly. Signal output from the bulk photodetector is digitized and assigned to the corresponding pixel location illuminated by the laser spot. The target and detector assembly are stationary while the laser spot is scanned in a programmed pattern. Areas up to 16 cm2 are scanned at a rate of 5.1 x iO pixels/s with 12 bits gray level detection range per pixel. Typically, 1024 x 1024 pixels are captured in less than 30 seconds and stored for display, processing or archiving. Spatial resolution for image reconstruction is a function of laser spot size and has been demonstrated to 3 pm. Rescan and arbitrary laser spot positioning is accomplished to within Biological, biophysical, clinical instrumentation and optical and computer engineering applications of this technology are broad. SLI methods have been developed for quantitative densitometric analysis of electrophoretic gels, thin-layer chromatography plates and autoradiographic materials generally used in molecular biology research. These applications are not well suited for conventional scanning densitometry, particularly when translucent materials such as nylon or nitrocellulose transfer membranes are used because of loss in resolution due to scattering. The SLI spatial resolution is not affected significantly by such scatter. Other quantitative analysis such as measurement of immunofluorescent and immunochemical staining of cells from blood samples and tissue sections can be performed with the SLI device. These applications are poorly suited for traditional flow cytometry, which requires dispersal of individual cells from the tissue. In addition to imaging, the SLI device is capable of detecting and analyzing the occurrence of very rare events. It is routinely capable of scanning a large transparent or translucent target, detecting submicron particles and recording the position of the particles to within approximately 1 pm. The system will detect and locate a single submicron particle placed anywhere on a 800 mm2 surface, a task analogous to locating a standard typewritten character placed on a football field, within 30 seconds.

Enhanced SV40 Immortalization of Primary Human Epidermal Cells Following Phorbol Ester Dependent EBV Transformation

The susceptibility of freshly isolated human epithelial cells to simian virus 40 (SV40) transformation is very low (1–5). It has been difficult to quantitate the sensitivity of these cells to SV40 transformation, since the end point is indefinite life span in vitro. None the less, the phenomenon is considered to be a relatively rare event, which may occur in 1 to 10% of cultures inoculated, and with substantially lower frequency within any single culture population.