Craig Dees

SIMULTANEOUS TWO-PHOTON ACTIVATION OF TYPE-I PHOTODYNAMIC THERAPY AGENTS

The excitation and emission properties of several psoralen derivatives are compared using conventional single‐photon excitation and simultaneous two‐photon excitation (TPE). Two‐photon excitation is effected using the output of a mode‐locked titanium: sapphire laser, the near infrared output of which is used to promote non‐resonant TPE directly. Specifically, the excitation spectra and excited‐state properties of 8‐methoxypsoralen and 4′‐aminomethyl‐4,5,8‐trimethylpsoralen are shown to be equivalent using both modes of excitation. Further, in vitro feasibility of two‐photon photodynamic therapy (PDT) is demonstrated using Salmonella typhimurium. Two‐photon excitation may be beneficial in the practice of PDT because it would allow replacement of visible or UV excitation light with highly penetrating, nondamag‐ing near infrared light and could provide a means for improving localization of therapy. Comparison of possible laser excitation sources for PDT reveals the titanium: sapphire laser to be exceptionally well suited for nonlinear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate that together provide efficient PDT activation and greatly reduced potential for biological damage

DDT mimicks estradiol stimulation of breast cancer cells to enter the cell cycle

Estrogens play a critical role in the etiology of found breast cancer. Estradiol promotes the growth of breast cancer cells in vivo and in vitro. Exogenous estrogens in both the environment and in the human diet increase the growth of breast cancer cells in vitro. A role for xenoestrogens in breast cancer etiology has been proposed but remains controversial. We examined the effects of the xenoestrogenic pesticide 1,1,1‐trichloro‐2,2‐bis(chlorophenyl)ethane (DDT) on estrogen‐receptor (ER)‐positive MCF‐7 and T‐47D human breast cancer cells as well as on ER‐negative HS 578Bst breast cancer cells and rat liver cells. Estradiol and DDT were found to increase the growth of MCF‐7 cells in the presence of insulin. The activity of cyclin‐dependent kinase (Cdk)2 increased in growth‐arrested T‐47D and MCF‐7 cells treated with β‐estradiol or DDT. The steroidal antiestrogen ICI 182,780 prevented both growth and Cdk2 activation induced by estradiol or DDT. Increased phosphorylation of Cdk2 and the retinoblastoma protein (pRb1O5) was observed in ER‐positive cells treated with DDT or estradiol. Cdk2 activity was not affected by DDT or estradiol in ER‐negative HS 578Bst breast cancer cells or in rat liver epithelial cells. Cyclin D1 protein synthesis was increased by DDT and estradiol in MCF‐7 cells. DDT and estradiol‐induced ER‐dependent transcriptional activation of estrogen response elements (EREs) in stably transfected MVLN cells, and ERE activation by low doses of DDT was increased by insulin. These findings suggest that DDT can stimulate breast cancer cells to enter into the cell cycle by directly affecting key regulatory elements. The relative potency of DDT in inducing cell‐cycle progression appears to be only 100–300 times less than that of estradiol when measured in the presence of insulin. Therefore, the cancer risks associated with DDT exposure may be greater than first thought, especially when additional mitogenic stimuli are present. Mol. Carcinog. 18:107–114, 1997.

Treatment of Murine Cutaneous Melanoma with Near Infrared Light

Treatment of cutaneous melanoma (M‐3 and B16‐F10 implanted in mice) with rapidly‐scanned, tightly‐focused near infrared light elicits selective destruction of tumor tissue. A single laser treatment yielded complete eradication in >90% of B16‐F10 tumors with thicknesses of approximately 3 mm; amelanotic M‐3 tumors proved less responsive (ca 25% clearance rate). In addition to local tumor destruction, laser treatment of B16‐F10 tumors in immunocompetent mice stimulated enhanced cytokine levels (interleukin‐2 and interleukin‐10) within treated tumor tissues and rejection of tumor cells upon a subsequent challenge dose. Such an antitumor immune response may lead to improved outcomes at both the treatment site and at sites of distant metastasis.

Effects of 60-Hz fields, estradiol and xenoestrogens on human breast cancer cells

If exposure to xenoestrogens or electromagnetic fields (EMFs) such as 60 Hz contributes to the etiology of breast cancer, it is likely that they must stimulate the growth of breast cells, damage genetic material or enhance the effects of other mitogenic or mutagenic agents (co-promotion). Therefore, the ability of xenoestrogens or exposure to 60-Hz fields to stimulate the entry of growth-arrested human breast cancer cells into the cell cycle was determined using cyclin-dependent kinase 2 (Cdk2) activity, synthesis of cyclin D1 and cdc2 activity. Exposure of estrogen receptor-positive MCF-7 or T-47D cells to estrogen and xenoestrogens (DDT and Red No. 3) increased Cdk2 and cyclin B1-cdc2 activity and cyclin D1 synthesis. Exposure of breast cancer cells to 12 mG or 1 or 9 G electromagnetic fields at 60 Hz failed to stimulate Cdk2 or cyclin B1-cdc2 activity or cyclin D1 synthesis. Simultaneous co-exposure of cells to 60-Hz fields and chemical promoters did not enhance Cdk2 activation above the levels produced by the chemical promoter alone. Estrogen and xenoestrogens also stimulated binding of the estrogen receptor to the estrogen receptor element but the EMF did not. Phorbol 12-myristate 13-acetate (PMA) induced phosphorylation of p53 and pRb1O5 in MCF-7 cells, but EMF exposure had no effect. DNA-damaging chemotherapeutic agents and Red Dye No. 3 were found to increase p53 site-specific DNA binding in breast cancer cells, but EMF exposure did not. Differential display analysis failed to detect any effect of EMF exposure on gene expression in MCF-7 cells, whereas the effects of estradiol were detected. These studies suggest that estrogen and xenoestrogens stimulate growth-arrested breast cancer cells to enter the growth cycle, but EMF exposure does not. Site-specific p53-DNA binding was increased in MC F-7 cells treated with DNA-damaging agents, but not by EMF exposure. EMF exposure does not appear to act as a promoter or DNA-damaging agent for human breast cancer cells in vitro.

An advanced bioprocessing concept for the conversion of waste paper to ethanol : scientific note

ConclusionsWaste paper is a plentiful and low-cost lignocellulosic feed material that may represent the most direct way to penetrate the market with an advanced bioprocessing system. Innovative bioprocessing concepts integrated into such a system for the production of ethanol should be economically viable. Several of the proposed processing advances for such a system have only been studied on a laboratory scale, so a more thorough process development and scale-up effort will be required.

The mitogenic potential of trichloroethylene in B6C3F1 mice

Male and female B6C3F1 mice were given TCE by gavage for 10 days. No histopathologic changes in the livers from control male and female mice were found. Moderate changes around central veins were noted in male or female mice that received 1000 mg/kg/body weight TCE. Histopathological changes included an increase in cytoplasmic eosinophilic staining and apoptosis around the central veins. Mitosis and DNA synthesis were examined using incorporation of [3H]thymidine into liver cells. Incorporation of [3H]thymidine was significantly increased in the DNA of animals receiving TCE. Total liver DNA extracted from TCA-treated mice was not significantly different than those of control groups. Autoradiographic examination of liver sections showed that the incorporation of label in control animals was primarily in perisinusoidal cells. The majority of radiolabel incorporation in TCE-treated mice was found in intermediate zone cells that appeared to be mature hepatocytes. No outstanding differences in the distribution of radiolabel in the liver from male or female mice were noted. When incorporation of [3H]thymidine was quantified by enumeration of labeled hepatocytes following autoradiography, incorporation of the radiolabel into hepatocytes increased proportionally to the applied dose of TCE, but did not increase in peri-sinusoidal cells. Increased mitotic figures in intermediate zone cells resembling mature hepatocytes were found in all mice treated with TCE. These results suggest that liver cell DNA synthesis and mitosis are stimulated by TCE and that these effects may be in part responsible for transformation of liver cells in these mice.

Characterization of the cellulose-degrading bacterium NCIMB 10462

The gram-negative cellulase-producing bacterium NCIMB 10462 has been previously namedPseudomonas fluorescens subsp. or var.cellulosa. Because of renewed interest in cellulose-degrading bacteria for use in the bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its true metabolic potential. Metabolic and physical characterization of NCIMB 10462 revealed that this is an alkalophilic, nonfermentative, gram-negative, oxidase-positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium has few characteristics consistent with a classification ofP. fluorescens and a very low probability match with the genusSphingomonas. However, total lipid analysis did not reveal that any sphingo-lipid bases are produced by this bacterium. NCIMB 10462 grows best aerobically, but also grows well in complex media under reducing conditions. NCIMB 10462 grows slowly under anaerobic conditions on complex media, but growth on cellulosic media occurred only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIMB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37°C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is its ability to degrade cellulose, we suggest that it be calledPseudomonas cellulosa.

Imaging photosensitizer distribution and pharmacology using multiphoton microscopy

Multiphoton microscopy is a powerful tool for imaging sub- cellular distribution of luminescent compounds present in living cells. We have used this tool to study the distribution and pharmacology of photosensitizers in tissue and tissue culture. Murine hepatoma tumor cells dosed with a photosensitizer were briefly photoactivated, then imaged for periods up to several hours. Using the photosensitizer Rose Bengal with green light activation, nearly immediate photolytic release of lysosomal enzymes resulted in catastrophic cell destruction within 5 - 30 minutes. The magnitude and rapidity of this response is markedly different than that observed with other photosensitizer agents, and is consistent with in vivo studies illustrating that Rose Bengal is capable of causing extremely rapid destruction of treated tumors.

Trichloroacetate stimulation of liver DNA synthesis in male and female mice

Male and female B6C3F1 mice were given trichloroacetate (TCA) by gavage for 11 days. Livers from untreated male and female mice were unremarkable by histopathologic examination. In livers from mice receiving 1000 mg/kg body weight, the centrolobular hepatic cords showed slight changes, which included increased eosinophilic staining and rare apoptosis. Areas in the intermediate zone were noted where the architecture of the liver hepatic cords was subtly changed. The changes in cord architecture seemed to define nodular areas where cellular proliferation in animals treated with TCA had occurred. No histopathologic differences were noted between the livers of treated or control, male and female animals. Mitosis and DNA synthesis were examined using incorporation of [3H]thymidine into liver cells. [3H]Thymidine incorporation into extracted liver DNA of animals receiving TCA was significantly increased over controls in all treatment groups. Autoradiographic examination of liver sections showed that the incorporation of label in control animals was predominantly in peri-sinusoidal cells, whereas the majority of radiolabel incorporation in TCA-treated animals was found in intermediate zone cells that appeared to be mature hepatocytes. No outstanding differences in the distribution of radiolabel in the liver sections from male or female mice were noted. When incorporation of [3H]thymidine was quantified by enumeration of labeled liver cells following autoradiography, incorporation of the radiolabel into hepatocytes increased with the dose of TCA given but there was no increase in radiolabel in peri-sinusoidal cells. Increased mitotic figures in intermediate zone cells resembling mature hepatocytes were noted in all mice treated with TCA. These results suggest that increased DNA synthesis and mitosis may contribute tumorigenesis by TCA.

Stabilization of the p53 gene product in two bovine leukemia virus infected cell lines

Fetal lamb kidney cells (FLK) and bat lung (BAT2CL6) cells that continuously produce bovine leukemia virus (BLV) were found to cause malignant tumors in nude mice. Uninfected bat lung cells (Tb 1 Lu) produced a small benign neoplasm. Pulse chase studies showed that the p53 gene product in BAT2CL6 cells was more stable compared with p53 in Tb 1 Lu cells. Mono-clonal antibody studies suggested that a mutant form of the p53 protein was produced in BLV-infected cells. Heteroduplex mapping studies of the p53 gene from BLV-infected cells also suggested that a mutation in p53 had occurred. Stabilization of the p53 gene product in BLV-infected cells may contribute to the progression of tumor virulence.