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Background & Aims: APC gene mutation is an early alteration in most colorectal tumors. In an attempt to determine its role in tumor development, we asked whether reintroducing wild-type APC into colorectal cancer cells with mutant APC affected cell cycle progression. Methods: Using transient transfection, a plasmid containing the APC complementary DNA and DNA encoding the green fluorescent protein was expressed in SW480 cells. In addition, several other constructs were co-expressed with APC to determine their combined effects. Results: We report that colorectal cancer cell lines transfected with wild-type APC arrest in the G 1 - phase of the cell cycle and that this arrest is abrogated by cotransfecting constitutively active β-catenin or cyclin D1 and cMYC together. This APC-induced cell cycle arrest involves the disruption of β-catenin–mediated transcription and depends on components of the G 1 /S regulatory machinery, as overexpression of E1a or E2F-1 , - 2 , or - 3 overrides the G 1 arrest. Consistent with this, APC transfection inhibits RB phosphorylation and reduces levels of cyclin D1. Conclusions: Our results suggest that APC functions upstream of RB in the G 1 /S regulatory pathway, cyclin D1 and cMYC affect APC-mediated arrest equivalently to oncogenic β-catenin , and most colon tumors disrupt control of G 1 /S progression by APC mutation. GASTROENTEROLOGY 2002;123:751-763

Bone marrow progenitor cells develop into mature tissue myeloid cells under the influence of colony-stimulating factors. Cytokines that are elevated post-thermal injury have been shown to influence this process. We hypothesize that thermal injury alters myelopoiesis at the level of the progenitor cell. These differences should be visible after in vitro cultures that include colony-stimulating factors. Prior to culture, bone marrow at postburn day 1 (PBD1) was assessed for cell surface markers and the levels of myeloid progenitors. After culture in granulocyte/macrophage-stimulating colony-stimulating factor, the cell surface markers of the cultured cells were determined. PBD1 marrow from thermally injured rats had more progenitor cells responsive to granulocyte/macrophage-stimulating colony-stimulating factor than did sham. Cultured PBD1 marrow produced more CD90br MYbr CD45dim CD4− MHCII− CD11bdim eosinophils than did sham. Cultured bone marrow from thermally injured animals produces myeloid cells with an altered phenotype. Similar changes in myelopoiesis may take place in vivo.

We propose that some of the critical molecules involved in the transfer of melanosomes from melanocytes to keratinocytes include plasma membrane lectins and their glycoconjugates. To investigate this mechanism, co-cultures of human melanocytes and keratinocytes derived from neonatal foreskins were established. The process of melanosome transfer was assessed by two experimental procedures. The first involved labeling melanocyte cultures with the fluorochrome CFDA. Labeled melanocytes were subsequently co-cultured with keratinocytes, and the transfer of fluorochrome assessed visually by confocal microscopy and quantitatively by flow cytometry. The second investigative approach involved co-culturing melanocytes with keratinocytes, and processing the co-cultures after 3 days for electron microscopy to quantitate the numbers of melanosomes in keratinocytes. Results from these experimental approaches indicate significant transfer of dye or melanosomes from melanocytes to keratinocytes that increased with time of co-culturing. Using these model systems, we subsequently tested a battery of lectins and neoglycoproteins for their effect in melanosome transfer. Addition of these selected molecules to co-cultures inhibited transfer of fluorochrome by approximately 15-44% as assessed by flow cytometry, and of melanosomes by 67-93% as assessed by electron microscopy. Therefore, our results suggest the roles of selected lectins and glycoproteins in melanosome transfer to keratinocytes in the skin.

Tyrosinase related protein-1 (TRP-1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.

UV radiation is an important etiologic factor for skin cancer, including melanoma. Constitutive pigmentation and the ability to tan are considered the main photoprotective mechanism against sun-induced carcinogenesis. Pigmentation in the skin is conferred by epidermal melanocytes that synthesize and transfer melanin to keratinocytes. Therefore, insuring the survival and genomic stability of epidermal melanocytes is critical for inhibiting photocarcinogenesis, particularly melanoma, the most deadly form of skin cancer. The paracrine factors α-melanocortin and endothelin-1 are critical for the melanogenic response of cultured human melanocytes to UV radiation. We report that α-melanocortin and endothelin-1 rescued human melanocytes from UV radiation–induced apoptosis and reduced DNA photoproducts and oxidative stress. The survival effects of α-melanocortin and endothelin-1 were mediated by activation of the melanocortin 1 and endothelin receptors, respectively. Treatment of melanocytes with α-melanocortin and/or endothelin-1 before exposure to UV radiation activated the inositol triphosphate kinase-Akt pathway and increased the phosphorylation and expression of the microphthalmia-related transcription factor. Treatment with α-melanocortin and/or endothelin-1 enhanced the repair of cyclobutane pyrimidine dimers and reduced the levels of hydrogen peroxide induced by UV radiation. These effects are expected to reduce genomic instability and mutagenesis.

7H-Dibenzo[c,g]carbazole (DBC), an N-heterocyclic aromatic hydrocarbon, is cytotoxic and carcinogenic in rodent liver. While DBC leads to necrotic lesions in the liver, the induction of apoptosis by DBC has not been investigated. The focus of this study was to determine the degree to which apoptosis and necrosis contributed to DBC cytotoxicity in a human hepatoma cell line (HepG2). To determine if these effects were unique to DBC, the results were compared to another hepatotoxin, aflatoxin B(1) (AFB(1)). DBC produced a distinct biphasic LDH release curve within 24 h of exposure. During the same time period lower concentrations of DBC (

T tremendous production and use the carbon-chloroform extract, or CCE. of organic chemicals in contempoA second extraction, by alcohol, yields rary society have resulted in new and the carbon-alcohol extract, or CAE. challenging problems for water purBased on the chloroform extract, veyors and control agencies. To define USPBS adopted a recommended standthe problems, field and laboratory ard for drinking water of not more methods must be available to assess orthan 0.2 ppm of CCE.3 No standard ganic-chemical concentrations. Until has been set for the CAE. recently, however, it was difficult-if Unfortunately, the CAM is not free not impossible-to measure the confrom inherent limitations. Neither centrations of most of the organic adsorption nor desorption efficiencies chemicals that might be present in are known for the multitude of organic water. Several important analytic adcompounds found in water. In studies vances have changed the situation. of these efficiency factors, only a limited One of these is the carbon adsorption number of organic compounds have method (CAM); another is improved been used. In one study," phenol adinstrumentation, such as the gas-liquid sorption on activated carbon varied chromatograph. The carbon adsorpfrom 30 to 99 per cent; desorption or tion method permits the collection of extraction, 61 to 77 per cent; and relatively large samples, whereas gasoverall recovery, 19 to 77 per cent. liquid chromatography allows identifiIn a study by Hoak," phenol adsorption cation and quantitative estimation of on carbon was uniformly high (not less extremely small amounts of individual than 94 per cent), but recovery from chemicals extracted from conventionalthe carbon was as low as 22 per cent. size samples. Hoak showed that chemical changes in The carbon adsorption method was the adsorbed phenol could markedly developed by USPHS in 1951.1,2 In affect the apparent efficiency. In an this procedure the water to be samarticle on the recovery of chlorinated pled is pumped through a column conpesticides," Rosen and Middleton containing activated carbon, which adsorbs, eluded that It ••• results may be low by hence concentrates, the organic suba factor of two." More recent studies 7 stances in the water. The extraction, have shown recoveries of toxaphene by chloroform, of the adsorbed organic and the gamma isomer of benzene hexasubstances from the carbon produces chloride to be about 50 per cent.

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