Tumor suppressor and oncogene actions of TGFbeta1 occur early in skin carcinogenesis and are mediated by Smad3.

Interactions between TGFbeta1 and ras signaling pathways play an important role in cancer development. Here we show that in primary mouse keratinocytes, v-ras(Ha) does not block the early biochemical events of TGFbeta1 signal transduction but does alter global TGFbeta1 mediated gene expression in a gene specific manner. Expression of Smad3 dependent TGFbeta1 early response genes and the TGFbeta1 cytostatic gene expression response were not altered by v-ras(Ha) consistent with an intact TGFbeta1 growth arrest. However, TGFbeta1 and v-ras(Ha) cause significant alteration in genes regulating matrix remodeling as the TGFbeta1 induction of extracellular matrix genes was blocked by v-ras(Ha) but specific matrix proteases associated with cancer progression were elevated. Smad3 deletion in keratinocytes repressed normal differentiation maker expression and caused expression of Keratin 8 a simple epithelial keratin and marker of malignant conversion. Smad3 was required for the TGFbeta1 cytostatic response in v-ras(Ha) keratinocytes, but also for protease induction, keratinocyte attachment and migration. These results show that pro-oncogenic activities of TGFbeta1 can occur early in carcinogenesis before loss of its tumor suppressive function and that selective regulation rather than complete inactivation of Smad3 function may be crucial for tumor progression.

Chemical mixture toxicology: from descriptive to mechanistic, and going on to in silico toxicology.

Because of the pioneering vision of certain leaders in the biomedical field, the last two decades witnessed rapid advances in the area of chemical mixture toxicology. Earlier studies utilized conventional toxicology protocol and methods, and they were mainly descriptive in nature. Two good examples might be the parallel series of studies conducted by the U.S. National Toxicology Program and TNO in The Netherlands, respectively. As a natural course of progression, more and more sophistication was incorporated into the toxicology studies of chemical mixtures. Thus, at least the following seven areas of scientific achievements in chemical mixture toxicology are evident in the literature: (a) the application of better and more robust statistical methods; (b) the exploration and incorporation of mechanistic bases for toxicological interactions; (c) the application of physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling; (d) the studies on more complex chemical mixtures; (e) the use of science-based risk assessment approaches; (f) the utilization of functional genomics; and (g) the application of technology. Examples are given for the discussion of each of these areas. Two important concepts emerged from these studies and they are: (1) dose-dependent toxicologic interactions; and (2) "interaction thresholds". Looking into the future, one of the most challenging areas in chemical mixture research is finding the answer to the question "when one tries to characterize the health effects of chemical mixtures, how does one deal with the infinite number of combination of chemicals, and other possible stressors?" Undoubtedly, there will be many answers from different groups of researchers. Our answer, however, is first to focus on the finite (biological processes) rather than the infinite (combinations of chemical mixtures and multiple stressors). The idea is that once we know a normal biological process(es), all stimuli and insults from external stressors are merely perturbations of the normal biological process(es). The next step is to "capture" the biological process(es) by integrating the recent advances in computational technology and modern biology. Here, the computer-assisted Reaction Network Modeling, linked with PBPK modeling, offers a ray of hope to dealing with the complex biological systems.

Gene expression patterns as potential molecular biomarkers for malignant transformation in human keratinocytes treated with MNNG, arsenic, or a metal mixture.

In previous studies, treatment with 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) enhanced malignant transformation of immortal human epidermal (RHEK-1) keratinocytes. In contrast, arsenic (As) alone or in a mixture of As, cadmium (Cd), chromium (Cr), and lead (Pb) inhibited this process. Microarray analysis showed unique gene expression patterns in RHEK-1 exposed to MNNG, As, or the metal mixture. From this analysis, we have selected 16 genes potentially involved in the enhancement or inhibition of transformation. These 16 genes, nine (IFN inducible protein 9-27, MAA A32, CCLB protein, integrin beta4, XRCC1, K8, K18, MT3, MAPKK6) of which were altered in a chemical-specific manner and seven (MIC1, bikunin, MTS1, BMP4, RAD23A, DOC2, vimentin) of which were commonly affected by the MNNG and As or mixture treatments, were examined for expression in detail by real-time RT-PCR. Qualitatively, both microarray and real-time RT-PCR analyses gave comparable results for 15 of 16 genes, i.e., genes were consistently induced or suppressed under the different treatment regimens when measured by either technique. Of the seven genes altered in their expression by multiple chemical treatments, five showed patterns consistent with a role in the transformation process, i.e., they were oppositely regulated in MNNG-transformed RHEK-1 cells (designated as OM3) as compared to the nonmalignant As- and mixture-exposed cells. Through time-course studies, we also identified markers whose expression correlates with acquisition of transformation-associated characteristics in OM3. Identification of a battery of genes altered during progressive transformation of RHEK-1 should aid in developing a mechanistic understanding of this process, as well as strengthening the utility of these genes as biomarkers.

Characterization of gene expression changes associated with MNNG, arsenic, or metal mixture treatment in human keratinocytes: application of cDNA microarray technology.

The identification of molecular markers related to critical biological processes during carcinogenesis may aid in the evaluation of carcinogenic potentials of chemicals and chemical mixtures. Work from our laboratory demonstrated that a single treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhanced spontaneous malignant transformation of the human keratinocyte cell line RHEK-1. In contrast, chronic low-level exposure of cells to arsenic alone or in a mixture containing arsenic, cadmium, chromium, and lead inhibited malignant conversion. To identify changes in gene expression that influence these different outcomes, cDNA microarray technology was used. Analysis of multiple human arrays in MNNG-transformed RHEK-1 cells, designated OM3, and those treated with arsenic or the arsenic-containing metal mixture showed unique patterns of gene expression. Genes that were overexpressed in OM3 included oncogenes, cell cycle regulators, and those involved in signal transduction, whereas genes for DNA repair enzymes and inhibitors of transformation and metastasis were suppressed. In arsenic-treated cells, multiple DNA repair proteins were overexpressed. Mixture-treated cells showed increased expression of a variety of genes including metallothioneins and integrin 4. These cells showed decreased expression of oncogenes, DNA repair proteins, and genes involved in the mitogen-activated protein kinase pathway. For comparison we are currently analyzing gene expression changes in RHEK-1 cells transformed by other means. The goal of these studies is to identify common batteries of genes affected by chemical modulators of the carcinogenic process. Mechanistic studies may allow us to correlate alterations in their expression with sequential stages in the carcinogenic process and may aid in the risk assessment of other xenobiotics.

Experimental designs for mixtures of chemicals along fixed ratio rays.

Experimental design is important when studying mixtures/combinations of chemicals. The traditional approach for studying mixtures/combinations of multiple chemicals involves response surface methodology, often supported by factorial designs. Although such an approach permits the investigation of both the effects of individual chemicals and their interactions, the number of design points needed to study the chemical mixtures becomes prohibitive when the number of compounds increases. Fixed ratio ray designs have been developed to reduce the amount of experimental effort when interest can be restricted to a specific ray. We focus on the design and analysis issues involved in studying mixtures/combinations of compounds along fixed ratio rays of the compounds. To obtain the inference regarding the interactions among the compounds, we show that the only data required are those along the fixed ratio ray.