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High-content cell imaging based on fluorescent protein reporters has recently been used to track the transcriptional activities of multiple genes under different external stimuli for extended periods. This technology enhances our ability to discover treatment-induced regulatory mechanisms, temporally order their onsets and recognize their relationships. To fully realize these possibilities and explore their potential in biological and pharmaceutical applications, we introduce a new data processing procedure to extract information about the dynamics of cell processes based on this technology. The proposed procedure contains two parts: (1) image processing, where the fluorescent images are processed to identify individual cells and allow their transcriptional activity levels to be quantified; and (2) data representation, where the extracted time course data are summarized and represented in a way that facilitates efficient evaluation. Experiments show that the proposed procedure achieves fast and robust image segmentation with sufficient accuracy. The extracted cellular dynamics are highly reproducible and sensitive enough to detect subtle activity differences and identify mechanisms responding to selected perturbations. This method should be able to help biologists identify the alterations of cellular mechanisms that allow drug candidates to change cell behavior and thereby improve the efficiency of drug discovery and treatment design.

Since its origin in the mid-1990s, gene expression profiling by microarray has become a productive and useful tool in basic science and preclinical research. Current dual-color, high-density cDNA oligo arrays contain 60-mer detectors for the whole human genome. With this powerful technology, expression of RNA samples from cell lines or tissue can be assessed, revealing specific gene expression signatures. The technique includes three major steps: (1) isolation and purification of RNA from cells or tissues, (2) labeling of total RNA, and (3) hybridization with Agilent cDNA microarrays. Conveniently, this technique can be performed with as little as 50 ng of purified total RNA; however, it is important to keep in mind that the quality of the RNA template, namely the level of sample degradation and the presence of contaminants that are carried over from the starting material or introduced during RNA isolation, can significantly impact the efficiency of the labeling reaction and the reliability of the hybridization. In this chapter, the details of each step of this technique are explained thoroughly, while highlighting the key issues that can prevent a failed hybridization.

Multimeric ligands are ligands that contain multiple binding domains that simultaneously target multiple cell-surface proteins. Due to cooperative binding, multimeric ligands can have high avidity for cells (tumor) expressing all targeting proteins and only show minimal binding to cells (normal tissues) expressing none or only some of the targets. Identifying combinations of targets that concurrently express in tumor cells but not in normal cells is a challenging task. Here, we describe a novel approach for identifying such combinations using genome-wide gene expression profiling followed by immunohistochemistry. We first generated a database of mRNA gene expression profiles for 28 pancreatic cancer specimens and 103 normal tissue samples representing 28 unique tissue/cell types using DNA microarrays. The expression data for genes that encode proteins with cell-surface epitopes were then extracted from the database and analyzed using a novel multivariate rule-based computational approach to identify gene combinations that are expressed at an efficient binding level in tumors but not in normal tissues. These combinations were further ranked according to the proportion of tumor samples that expressed the sets at efficient levels. Protein expression of the genes contained in the top ranked combinations was confirmed using immunohistochemistry on a pancreatic tumor tissue and normal tissue microarrays. Coexpression of targets was further validated by their combined expression in pancreatic cancer cell lines using immunocytochemistry. These validated gene combinations thus encompass a list of cell-surface targets that can be used to develop multimeric ligands for the imaging and treatment of pancreatic cancer.

The inhibitory effect of caveolin on the cellular response to growth factor stimulation is well established. Given the significant overlap in signaling pathways involved in regulating cell proliferation and stress responsiveness, we hypothesized that caveolin would also affect a cell's ability to respond to environmental stress. Here we investigated the ability of caveolin-1 to modulate the cellular response to sodium arsenite and thereby alter survival of the human cell lines 293 and HeLa. Cells stably transfected with caveolin-1 were found to be much more sensitive to the toxic effects of sodium arsenite than either untransfected parental cells or parental cells transfected with an empty vector. Unexpectedly, the caveolin-overexpressing cells also exhibited a significant activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which additional studies suggested was likely due to decreased neutral sphingomyelinase activity and ceramide synthesis. In contrast to its extensively documented antiapoptotic influence, the elevated activity of Akt appears to be important in sensitizing caveolin-expressing cells to arsenite-induced toxicity, as both pretreatment of cells with the PI3K inhibitor wortmannin and overexpression of a dominant-negative Akt mutant markedly improved the survival of arsenite-treated cells. This death-promoting influence of the PI3K/Akt pathway in caveolin-overexpressing cells appeared not to be unique to sodium arsenite, as wortmannin pretreatment also resulted in increased survival in the presence of H(2)O(2). In summary, our results indicate that caveolin-induced upregulation of the PI3K/Akt signaling pathway, which appears to be a death signal in the presence of arsenite and H(2)O(2), sensitizes cells to environmental stress.

Oxidative stress is believed to be an important factor in the development of age-related diseases, and studies in lower organisms have established links between oxidative stress tolerance and longevity. We have hypothesized that aging is associated with a reduced ability to mount acute host defenses to oxidant injury, which increases the vulnerability of aged cells to stress. We tested this hypothesis by using primary hepatocytes from young (4-6 months) and aged (24-26 months) rats. Old hepatocytes were more sensitive to H2O2-induced apoptosis than were young cells. Lower survival is associated with reduced activations of extracellular signal-regulated kinase (ERK) and Akt kinase, both of which protect against oxidant injury. That reduced ERK and Akt activities contribute to lower survival of aged cells was supported by additional findings. First, pharmacologic inhibition of ERK and Akt activation in young cells markedly increased their sensitivity to H2O2. Second, caloric restriction, which increases rodent life span and delays the onset of many age-related declines in physiologic function, prevented loss in ERK and Akt activation by H2O2 and enhanced survival of old hepatocytes to levels similar to those of young cells. Strategies aimed at boosting these host responses to acute oxidant injury could have significant anti-aging benefits.

All cells depend on sterols and isoprenoids derived from mevalonate (MVA) for growth, differentiation, and maintenance of homeostatic functions. In plants, environmental insults like heat and sunlight trigger the synthesis of isoprene, also derived from MVA, and this phenomenon has been associated with enhanced tolerance to heat. Here, we show that in human prostate adenocarcinoma PC-3M cells heat shock leads to activation of the MVA pathway. This is characterized by a dose- and time-dependent elevation in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) activity, enhanced sterol and isoprenoid synthesis, and increased protein prenylation. Furthermore, prenylation and subsequent membrane localization of Ras, a central player in cell signaling, was rapidly induced following heat stress. These effects were dose-dependent, augmented with repeated insults, and were prevented by culturing cells in the presence of lovastatin, a competitive inhibitor of HMGR. Enhanced Ras maturation by heat stress was also associated with a heightened activation of extracellular signal-regulated kinase (ERK), a key mediator of both mitogenic and stress signaling pathways, in response to subsequent growth factor stimulation. Thus, activation of the MVA pathway may constitute an important adaptive host response to stress, and have significant implications to carcinogenesis.

Phenylacetate (PA) and related aromatic fatty acids constitute a novel class of relatively nontoxic antineoplastic agents. These compounds induce tumor cytostasis and growth inhibition and differentiation of cancer cells, but little is known regarding the molecular events mediating these biological effects. Using human breast carcinoma MCF-7 cells as a model, we show here that PA-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 and dephosphorylation of the retinoblastoma protein (pRB). The induction of p21WAF1/CIP1 mRNA by PA was independent of the cellular p53 status. To directly assess the contribution of p21Waf1/Cip1 to PA-mediated cytostasis, we compared the effects of PA in parental MCF-7 cells and cells expressing reduced levels of p21Waf1/Cip1 protein (clones AS.3 and AS.4), accomplished through constitutive expression of antisense p21Waf1/Cip1 transcripts. In contrast to parental cells, AS.3 and AS.4 cells did not show reduced pRB phosphorylation following PA treatment, indicating that p21Waf1/Cip1 induction by PA is required for dephosphorylation (inactivation) of pRB, a known mediator of cell cycle control. A prominent role for p21Waf1/Cip1 in mediating PA-induced growth arrest was further supported by the demonstration that embryonal fibroblasts derived from a p21WAF1/CIP1 knockout mouse (p21-/- mouse embryonal fibroblasts) did not growth arrest following PA treatment, whereas PA effectively induced p21WAF1/CIP1 mRNA and growth inhibition of the wild-type mouse embryonal fibroblasts. Taken together, our findings strongly support a role for p21Waf1/Cip1 in the PA-mediated inhibition of cell growth.

Cytotoxic chemotherapies often give rise to multidrug resistance, which remains a major problem in cancer management. In pursuit of alternative treatments for chemoresistant tumor cells, we tested the response of multidrug-resistant (MDR) tumor cell lines to the aromatic fatty acids phenylacetate (PA) and phenylbutyrate (PB), two differentiation inducers currently in clinical trials. Both compounds induced cytostasis and maturation of multidrug-resistant breast, ovarian, and colon carcinoma cells with no significant effect on cell viability. In contrast to their poor response to doxorubicin, the MDR cells were generally more sensitive to growth arrest by PA and PB than their parental counterparts. The aromatic fatty acids, like the differentiation-inducing aliphatic fatty acid butyrate, up-regulated mdr-1 gene expression. However, while butyrate increased multidrug resistance, PA and PB potentiated the cytotoxic activity of doxorubicin against MDR cells. The latter was associated with time-dependent declines in glutathione levels and in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, the antioxidant enzymes implicated in cell resistance to free radical-based therapies. Taken together, our in vitro data indicate that PA and PB, differentiation inducers of the aromatic fatty acid class, may provide an alternative approach to the treatment of MDR tumors.

The aromatic fatty acid phenylacetate, a common metabolite of phenylalanine, shows promise as a relatively non-toxic drug for cancer treatment. This slowly metabolized fatty acid alters tumor cell lipid metabolism causing, among other effects, inhibition of protein prenylation critical to malignant growth. In pursuit of more potent analogues, we have examined the activity of related compounds against tumor cell lines established from patients with advanced prostatic carcinoma, glioblastomas, and malignant melanoma. Like phenylacetate, derivatives containing alpha-carbon or ring substitutions induced cytostasis and phenotypic reversion at non-toxic concentrations. Potency was correlated with the degree of calculated lipophilicity of the aromatic fatty acid, and the extent of inhibition of protein prenylation. Remarkably, a parallel cytostatic activity was reported in embryonic plant cells, which respond to phenylacetate and its analogues in the same concentration range and the same rank order of lipophilicity. These data suggest that phenylacetate and its analogues may act through common mechanisms to inhibit the growth of vastly divergent, undifferentiated cell types, and provide a basis for the development of new agents for the treatment of human malignancies.

Alterations in the expression of ras oncogenes are characteristic of a wide variety of human neoplasms. Accumulating evidence has linked elevated ras expression with disease progression and with failure of tumors to respond to conventional therapies, including radiotherapy and certain chemotherapies. These observations led us to investigate the response of ras-transformed cells to the differentiation-inducer phenylacetate (PA). Using gene transfer models, we show that PA caused cytostasis in ras-transformed mesenchymal cells, associated with increased expression of 2',5'-oligoadenylate synthetase, an enzyme implicated in negative growth control. PA also induced phenotypic reversion characterized by loss of anchorage-independent growth, reduced invasiveness and increased expression of collagen alpha type I, a marker of cell differentiation. The anti-tumor activity of PA was observed in cases involving either Ha- or Ki-ras and was independent of the mode of oncogene activation. Interestingly, in contrast to their relative resistance to radiation and doxorubicin, ras-transformed cells were significantly more sensitive to PA than their parental cells. The profound changes in tumor cell and molecular biology were associated with reduced isoprenylation of the ras-encoded p21. Our results indicate that PA can suppress the growth of ras-transformed cells, resistant otherwise to free-radical based therapies, through interference with p21ras isoprenylation, critical to signal transduction and maintenance of the malignant phenotype.

Differentiation inducers selected for their low cytotoxic and genotoxic potential could be of major value in chemoprevention and maintenance therapy. We focus here on phenylacetate, a naturally occurring plasma component recently shown to affect the growth and differentiation of established neoplasms in experimental models. The ability of phenylacetate to prevent carcinogenesis by the chemotherapeutic hypomethylating drug 5-aza-2'-deoxycytidine (5AzadC) was tested in vitro and in mice. Transient exposure of immortalized, but poorly tumorigenic ras-transformed 4C8 fibroblasts to 5AzadC resulted in neoplastic transformation manifested by loss of contact inhibition of growth, acquired invasiveness, and increased tumorigenicity in athymic mice. The latter was associated with elevation in ras expression and a decline in collagen biosynthesis. These profound phenotypic and molecular changes were prevented by a simultaneous treatment with phenylacetate. Protection from 5AzadC carcinogenesis by phenylacetate was: (a) highly efficient despite DNA hypomethylation by both drugs, (b) free of cytotoxic and genotoxic effects, (c) stable after treatment was discontinued, and (d) reproducible in vivo. Whereas athymic mice bearing 4C8 cells developed fibrosarcomas following a single i.p. injection with 5AzadC, tumor development was significantly inhibited by systemic treatment with nontoxic doses of phenylacetate. Phenylacetate and its precursor suitable for oral administration, phenylbutyrate, may thus represent a new class of chemopreventive agents, the efficacy and safety of which should be further evaluated.

Cinnamic acid, a naturally occurring aromatic fatty acid of low toxicity, has a long history of human exposure. We now show that cinnamic acid induces cytostasis and a reversal of malignant properties of human tumor cells in vitro. The concentration causing a 50% reduction of cell proliferation (IC50) ranged from 1 to 4.5 mM in glioblastoma, melanoma, prostate and lung carcinoma cells. Using melanoma cells as a model, we found that cinnamic acid induces cell differentiation as evidenced by morphological changes and increased melanin production. Moreover, treated cells had reduced invasive capacity associated with modulation of expression of genes implicated in tumor metastasis (collagenase type IV, and tissue inhibitor metalloproteinase 2) and immunogenicity (HLA-A3, class-I major histocompatibility antigen). Further molecular analysis indicated that the anti-tumor activity of cinnamic acid may be due in part to the inhibition of protein isoprenylation known to block mitogenic signal transduction. The results presented here identify cinnamic acid as a new member of the aromatic fatty acid class of differentiation-inducers with potential use in cancer intervention.

Phenylacetate, a natural metabolite of phenylalanine which was originally described as a plant growth hormone, has recently gained attention as a possible differentiation inducer for a variety of human tumor cell types. This interest prompted us to assess the ability of sodium phenylacetate (NaPA) to promote the differentiation of human neuroblastoma cells, both alone and in combination with retinoic acid (RA), a known inducer of neuroblastoma differentiation and maturation. Using the LA-N-5 cell line, we have determined that NaPA can stimulate the differentiation of neuroblastoma cells, as evidenced by dose-dependent inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity and reduction of N-myc expression. Furthermore, NaPA and RA synergized in inducing differentiation, in that combination treatment resulted in cessation of cell growth along with morphologic and biochemical changes indicative of the loss of malignant properties. We have determined that NaPA can markedly enhance mRNA levels of the nuclear RA receptor-beta (RAR beta) in LA-N-5 cells prior to morphologic or other phenotypic changes induced by this compound. This effect appeared to be distinct from the ability of NaPA to alter tumor cell lipid metabolism via inhibition of protein isoprenylation. Thus among its varied effects on LA-N-5 cells, NaPA appears to interact with the RA pathway at the nuclear level by up-regulating RAR beta expression.

The increasing incidence of melanoma and the poor responsiveness of disseminated disease to conventional treatments call for the development of new therapeutic approaches. Phenylacetate, a nontoxic differentiation inducer, can suppress the growth of other neuroectodermal tumors, i.e., gliomas, in laboratory models and in humans. This finding led us to explore the efficacy of phenylacetate and related aromatic fatty acids in melanoma. Phenylacetate and phenylbutyrate were found to a) induce selective cytostasis and maturation of cultured human melanoma cells, b) modulate the expression of genes implicated in tumor metastasis (type IV collagenase and tissue inhibitor of metalloproteinases-2) and immunogenicity (HLA class I); and c) enhance the efficacy of other agents of clinical interest, including retinoids, interferon-alpha, suramin, and 5-aza-2'-deoxycytidine. Reflecting on the phenotypic heterogeneity of melanoma, the degree of biologic alterations induced by phenylacetate/phenylbutyrate varied significantly among the tumor cell lines tested. Although losing invasive capacity and tumorigenicity in athymic mice, poorly differentiated cells exhibited only a marginal change in morphology, remained amelanotic, and resumed growth after treatment was discontinued. By contrast, treatment of melanoma cells that were in a more advanced stage of maturation resulted in profound alterations in cell growth, morphology, and pigmentation consistent with terminal differentiation. The in vitro antitumor activity was observed with nontoxic, pharmacologic concentrations of phenylacetate and phenylbutyrate, suggesting potential clinical use of these drugs in the treatment of melanomas.

Phenylacetate, a deaminated metabolite of phenylalanine, has been implicated in damage to immature brain in phenylketonuria. Because primary brain tumors are highly reminiscent of the immature central nervous system, these neoplasms should be equally vulnerable. We show here that sodium phenylacetate can induce cytostasis and reversal of malignant properties of cultured human glioblastoma cells, when used at pharmacological concentrations that are well tolerated by children and adults. Treated tumor cells exhibited biochemical alterations similar to those observed in phenylketonuria-like conditions, including selective decline in de novo cholesterol synthesis from mevalonate. Because gliomas, but not mature normal brain cells, are highly dependent on mevalonate for production of sterols and isoprenoids vital for cell growth, sodium phenylacetate would be expected to affect tumor growth in vivo while sparing normal tissues. Systemic treatment of rats bearing intracranial gliomas resulted in significant tumor suppression with no apparent toxicity to the host. The data indicate that phenylacetate, acting through inhibition of protein prenylation and other mechanisms, may offer a safe and effective novel approach to treatment of malignant gliomas and perhaps other neoplasms as well.

Sodium phenylacetate was found to affect the growth and differentiation of tumor cells in vitro at concentrations that have been achieved in humans with no significant adverse effects. Treatment of promyelocytic leukemia HL-60 cells resulted in the rapid decline of myc oncogene expression followed by growth arrest and granulocyte differentiation. Phenylacetate also induced highly efficient adipocyte conversion in immortalized mesenchymal C3H 10T1/2 cultures; yet, unlike the differentiating chemotherapeutic drug 5-aza-2'-deoxycytidine, phenylacetate did not cause neoplastic transformation in these susceptible cells. The results indicate that phenylacetate is both effective in inducing tumor cell maturation and free of cytotoxic and carcinogenic effects, a combination that warrants attention to its potential use in cancer intervention.

Any attempt to eradicate the heterogeneous cell population of a tumour mass would require the use of appropriate combination treatment protocols. The antitumour effects of interferon alpha (IFN alpha) in combination with AS2-1, the hydrolysis product of 3-phenylacetyl-amino-2,6-piperidinedione, were examined using several human tumour cell lines as a model. These included the malignant melanoma A375, adenocarcinoma of the prostate PC3 (hormone-insensitive bone metastasis), and the erythroleukaemia line K562. AS2-1 suppressed tumour growth through non-toxic mechanisms, with 1 mg/ml causing approximately 50% inhibition of the melanoma and prostate tumour cell proliferation. By contrast, primary normal human skin fibroblasts were significantly less sensitive to the antiproliferative effect of AS2-1. Suppression of tumour growth was seen also with AS2-1 treatment of the erythroleukaemia K562; in these cultures the drug also induced dose-dependent differentiation, as indicated by the increased haemoglobin production. Interestingly, addition of low doses of IFN alpha markedly enhanced the antitumour and differentiating effects observed with AS2-1. Treatment with 200-300 IU/ml of IFN (which caused about 20% inhibition of growth) together with 1 mg/ml of AS2-1 resulted in over 80% inhibition of the melanoma and prostate cancer cell proliferation, suggesting a synergistic activity of the two agents. This was substantiated by quantitative analysis of the differentiation induced in K562 erythroleukaemia. It appears, therefore, that IFN alpha and AS2-1 may act through synergistic mechanisms to effectively inhibit tumour growth and promote differentiation in a variety of human malignant cell lines.