Reversibility of progression of the transformed phenotype in Ad5-transformed rat embryo cells.

The carcinogenic process is extremely complex and is affected by diverse environmental and host factors. The mechanism for the gradual development of the transformed phenotype (a process termed "progression") was studied in type 5 adenovirus (Ad5)-transformed rat embryo cells. Progression was not correlated with major changes in the pattern of integration of viral DNA sequences. Instead, it was associated with an increased methylation of integrated viral sequences other than those corresponding to the E1 transforming genes of Ad5. A single exposure of progressed cells to the demethylating agent 5-azacytidine (Aza) resulted in a stable reversion to the unprogressed state of the original parental clone. A further selection of cells after growth in agar allowed the isolation of Aza-treated clones that had regained the progressed phenotype. These observations indicate that progression is a reversible process and suggest that progression may be associated with changes in the state of methylation of one or more specific genes.

Expression and function of the leucine zipper protein Par-4 in apoptosis.

The prostate apoptosis response-4 (par-4) gene was identified by differential screening for genes that are upregulated when prostate cancer cells are induced to undergo apoptosis. The par-4 gene is induced by apoptotic signals but not by growth-arresting, necrotic, or growth-stimulatory signals. The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain at its carboxy terminus. We have recently shown that the Par-4 protein binds, via its leucine zipper domain, to the zinc finger domain of Wilms' tumor protein WT1 (R. W. Johnstone et al., Mol. Cell. Biol. 16:6945-6956, 1996). In experiments aimed at determining the functional role of par-4 in apoptosis, an antisense par-4 oligomer abrogated par-4 expression and activator-driven apoptosis in rat prostate cancer cell line AT-3, suggesting that par-4 is required for apoptosis in these cells. Consistent with a functional role for par-4 in apoptosis, ectopic overexpression of par-4 in prostate cancer cell line PC-3 and melanoma cell line A375-C6 conferred supersensitivity to apoptotic stimuli. Transfection studies with deletion mutants of Par-4 revealed that full-length Par-4, but not mutants that lacked the leucine zipper domain of Par-4, conferred enhanced sensitivity to apoptotic stimuli. Most importantly, ectopic coexpression of the leucine zipper domain of Par-4 inhibited the ability of Par-4 to enhance apoptosis. Finally, ectopic expression of WT1 attenuated apoptosis, and coexpression of Par-4 but not a leucine zipperless mutant of Par-4 rescued the cells from the antiapoptotic effect of WT1. These findings suggest that the leucine zipper domain is required for the Par-4 protein to function in apoptosis.

Analysis of lectin receptors on rat embryo fibroblasts transformed by adenovirus 2.

Transformation of rat embryo fibroblasts by human adenovirus type 2 (Ad2) results in the production of a series of cell lines that cover a spectrum of malignancy from nontumorigenic to highly tumorigenic in a single species. A panel of plant lectins was used to study surface characteristics of these cell lines that might correlate with tumorigenicity. Because of the complex nature of lectin-cell surface interactions, a number of parameters were determined; they included numbers and densities of lectin receptors, binding affinities, and receptor mobilities. The lectins from Lens culinaris, Lotus tetragonolobus, and Ricinus communis were found to be the most useful for differentiating among the various Ad2-transformed cell lines. In general, the more tumorigenic cell lines were characterized by high numbers of lectin receptors, high percentages of lectin-binding cells, and heterogeneous distributions of receptors from cell to cell. In contrast, the nontumorigenic and the weakly tumorigenic cell lines were characterized by low numbers of lectin receptors present on a minority of cells within each population and a more homogeneous distribution of these receptors from cell to cell. These data demonstrate that lectins can identify surface properties that appear to correlate with malignant potential in the Ad2-transformed cell lines.

Effect of methyl methanesulfonate on type 5 adenovirus DNA integration and the phenotypic properties of cold-sensitive type 5 adenovirus-transformed cloned rat embryo fibroblast cells.

Pretreatment of a cloned rat embryo fibroblast cell line (CREF) with methyl methanesulfonate (MMS) prior to infection with a specific cold-sensitive type 5 adenovirus mutant, H5hr1, results in a unique carcinogen enhancement of transformation phenotype. MMS induces a dose-dependent increase in the absolute number of transformed foci in comparison with solvent-treated controls as well as an increase in transformation frequency when normalized for carcinogen-induced cell toxicity. To determine if the carcinogen enhancement of transformation phenotype was a consequence of the carcinogen altering the pattern of type 5 adenovirus (Ad5) DNA integration into the genome of CREF cells and/or if carcinogen treatment modified the phenotype of established H5hr1-transformed CREF cells, we have analyzed a series of single cell-derived H5hr1-transformed CREF cultures which were isolated from cultures pretreated with carcinogen-solvent or MMS prior to infection with H5hr1. Analysis of viral DNA integration by DNA filter-transfer hybridization (Southern blotting) indicated that MMS pretreatment did not increase the copy number of Ad5 DNA sequences which persisted in H5hr1-transformed clones or result in transformants which contained identical DNA restriction enzyme cleavage patterns. MMS-pretreated H5hr1-transformed clones also did not differ significantly from solvent-pretreated H5hr1-transformed clones in their ability to grow in agar, bind 125I-epidermal growth factor, or form tumors in athymic nude mice. MMS-pretreated H5hr1-transformed CREF clones retained a similar cold-sensitive negative regulation in the expression of the transformed cell phenotype as did H5hr1-transformed clones not exposed to carcinogens. These findings suggest that the unique carcinogen enhancement of transformation phenotype displayed by CREF cells pretreated with MMS prior to infection with H5hr1 does not result in transformants which either contain increased concentrations of Ad5 DNA or similar patterns of Ad5 DNA integration. Furthermore, carcinogen-pretreated H5hr1 transformants did not display novel phenotypes not expressed by cloned H5hr1-transformed CREF cell lines exposed to solvent prior to viral infection.

Wild-type adenovirus type 5 transforming genes function as transdominant suppressors of oncogenesis in mutant adenovirus type 5 transformed rat embryo fibroblast cells.

Transformation of cloned rat embryo fibroblast (CREF) cells with the host-range adenovirus type 5 (Ad5) mutant, H5hr1, results in transformants with a fibroblastic morphology which displays a cold-sensitive transformation phenotype and oncogenic potential in both nude mice and syngeneic rats. In contrast, wild-type (wt) Ad5 transformed CREF cells are epithelioid in morphology, temperature independent for transformation, and nontumorigenic. The present studies were conducted to analyze the contribution of the mutated E1A and E1B regions of H5hr1 in regulating the biological properties of H5hr1-transformed CREF cells. CREF cells were constructed which contain the mutated E1A and E1B transforming regions of H5hr1 and either a wt Ad5 E1A gene, a wt Ad5 E1B gene, or both a wt Ad5 E1A and a wt E1B gene. A wt Ad5 E1A gene was sufficient in reversing the cold-sensitive transformation phenotype. By using a wt Ad5 E1A gene under the transcriptional control of a dexamethasone-inducible mouse mammary tumor virus promoter, a direct suppressive effect of wt Ad5 E1A on colony formation in monolayer culture and agar growth of H5hr1-transformed cells was demonstrated. Expression of a wt Ad5 E1A, a wt Ad5 E1B, or both wt transforming genes in H5hr1-transformed CREF cells also suppressed oncogenicity. The ability or inability to form tumors in animals was found not to correlate with sensitivity to natural killer cell-mediated lysis. These results indicate that both the wt Ad5 E1A and wt Ad5 E1B genes can function as dominant suppressors of the oncogenic process when coexpressed in H5hr1-transformed CREF cells. This effect does not require large quantities of wt Ad5 E1A or E1B transforming proteins, nor is it directly related to the acquisition of a natural killer cell cytolysis-susceptible phenotype.

A link between ras and metastatic behavior of tumor cells: ras induces CD44 promoter activity and leads to low-level expression of metastasis-specific variants of CD44 in CREF cells.

The activated oncogene c-Ha-ras induces expression of the surface glycoprotein CD44 in cloned rat embryonic fibroblasts (CREF). Induction is transcriptional as shown by transient cotransfections of c-Ha-ras expression constructs and CD44 promoter reporter gene constructs and depends on the presence of an AP-1 binding site at position -110. Increased transcript levels for the standard isoform of CD44 (CD44s) are accompanied by the appearance of alternatively spliced RNAs and the synthesis of variants of CD44 (CD44v). These CD44v molecules differ from the standard type by the addition of sequences in the extracellular portion of the molecules. The occurrence of CD44v molecules in CREF cells upon induction of the CD44 promoter is probably due to leakiness of the splice control in these cells since stable transfection with c-Ha-ras does not alter the CD44v/total CD44 ratio. Upon ras overexpression, however, using an inducible mouse mammary tumor virus-ras construct, a transient increase of CD44v/total CD44 ratio of 3-4 has been determined suggesting that a burst of ras expression, in the genetic background of CREF cells, influences both promoter activity and splice control or accuracy. The expression of CD44v proteins is responsible for the metastatic potential in a variety of tumors (U. Günthert et al., Cell, 65: 13-24, 1991). Also in CREF cells expression of CD44v correlates with metastatic behavior, ras-transfected CREF cells are not only fully transformed but also give rise to metastatic spread as measured in the spontaneous metastasis assay. The adenoviral oncogene E1A counteracts ras-induced promoter function and, consequently, inhibits metastatic behavior without extinguishing transformation.

Distinct patterns of E-cadherin CpG island methylation in papillary, follicular, Hurthle's cell, and poorly differentiated human thyroid carcinoma.

Expression of the invasion/metastasis suppressor, E-cadherin, is diminished or lost in thyroid carcinomas. Yet, mutational inactivation of E-cadherin is rare. Herein, we show that this loss is associated with hypermethylation of the E-cadherin 5' CpG island in a panel of human thyroid cancer cell lines. This aberrant methylation is evident in 83% of papillary thyroid carcinoma, 11% of follicular thyroid carcinoma, 40% of Hurthle's cell carcinoma, and 21% of poorly differentiated thyroid carcinomas. Contrary to previous reports, the majority of these poorly differentiated thyroid carcinomas express E-cadherin, but often within the cytoplasm rather than at the cell surface. Together, our data indicate that the invasion/metastasis suppressor function of E-cadherin is frequently compromised in human papillary, Hurthle's cell, and poorly differentiated thyroid carcinoma by epigenetic and biochemical events.

Defining the critical gene expression changes associated with expression and suppression of the tumorigenic and metastatic phenotype in Ha-ras-transformed cloned rat embryo fibroblast cells.

Carcinogenesis requires a complex series of genetic changes often involving multiple oncogenes and the inactivation of multiple tumor-suppressor genes. We presently examined the effect of the Krev-1 tumor-suppressor gene on the tumorigenic and metastatic potential of Ha-ras-transformed cloned rat embryo fibroblast (CREF) cells. Ha-ras-transformed CREF cells are morphologically transformed and anchorage independent; produce reduced levels of nm23-H1 (a putative metastasis-suppressor gene product) and TIMP-1 (tissue inhibitor of metalloproteinase 1) transcripts and mRNA compared with CREF cells; produce increased levels of cripto, 94-kDa gelatinase/type IV collagenase (94-kDa GEL), osteopontin (OPN) and transin/stromelysin transcripts and mRNA compared with CREF cells; and are tumorigenic and metastatic in both nude mice and syngeneic rats. Ha-ras-transformed CREF cells coexpressing the Krev-1 gene display a reversion in cellular phenotype and gene expression to that of untransformed CREF cells. However, Ha-ras/Krev-1-coexpressing CREF cells retain, albeit with extended latency periods, both tumorigenic and metastatic potential that is not related directly to the final level of Ha-ras or Krev-1 mRNA or the Ha-ras p21 transforming protein. Development of metastatic potential is, however, directly correlated with a reduction in nm23-H1 and TIMP-1 transcription and mRNA levels and an enhanced expression of cripto, 94-kDa GEL, osteopontin and transin. In contrast, expression of additional tumor-suppressor genes, such as the RB gene and p53, or genes associated with tumorigenesis in other model systems, such as major excreted glycoprotein (MEP), 72-kDa gelatinase/type IV collagenase (72-kDa GEL), fibronectin (FIB), tenascin and intracellular adhesion molecule 1 (ICAM-1) is not altered in a consistent manner during in vitro transformation suppression or escape from tumorigenic and metastatic suppression. These results indicate that Krev-1 suppression of the Ha-ras-transformed/oncogenic phenotype is associated with a distinct program of gene expression changes manifested by altered rates of transcription and steady-state mRNA levels of specific oncogenic-suppressing and oncogenic-inducing genes. These data support a model of Ha-ras-induced metastasis in CREF cells that involves a direct modulation in the expression/suppression of specific combinations of oncogenic-suppressor genes and metastasis-promoting genes that are regulated coordinately in the process of tumor progression.

Elevated expression of eIF4E and FGF-2 isoforms during vascularization of breast carcinomas.

The translation initiation factor eIF4E is a novel protooncogene found over expressed in most breast carcinomas (Kerekatte et al., 1995), but the pathology where this elevation is initially manifested and its possible role in cancer progression are unknown. We report that eIF4E is markedly increased in vascularized malignant ductules of invasive carcinomas, whereas necrotic and avascular ductal carcinomas in situ display significantly lower levels. eIF4E facilitates the synthesis of FGF-2, a powerful tumor angiogenic factor. Conversely, reducing eIF4E with antisense RNA in MDA-435 cells suppresses their tumorigenic and angiogenic properties, consistent with loss of FGF-2 synthesis. These findings suggest a causal role for eIF4E in tumor vascularization.

SV-40 virus-induced neoplastic transformation of hamster brain cells in vitro. Studies with scanning and transmission electron microscopy.

Neoplastic transformation of one-day-old hamster brain cells was produced by infection with SV-40 virus and verified by phase contrast microscopy, growth in semisolid media, and intracranial tumor production after inoculation of the cells into other one-day-old hamsters. Transformed cells were studied by transmission and scanning electron microscopy. The numerous alterations in cell surface structure and in nuclear and cytoplasmic organization suggest a marked increase in cell metabolism and in the rate of mitosis and cell division. Cilia with a nine-to-zero pattern of microtubule doublets were present in cells with intermediate size filaments which stained for glial fibrillary acidic protein. The findings indicate that infection of one-day-old hamster brain cells in culture by SV-40 virus results in their transformation to a neoplastic state and the transformed cells are differentiating neoplastic astrocytes.

The influence of the presence of adenovirus 5 E1a and E1b sequences on the pathology of rat embryonic fibroblasts transfected with activated c-Ha-ras and v-ras.

We compared the pathology of two groups of tumors following implantation of cells enmeshed in alginate beads into the syngeneic rat. The first group of tumors was generated by implanting alginate beads containing cloned embryonic fibroblasts (CREF) that were transfected with activated c-Ha-ras (T24) and v-ras (pH1) (CREF tumors). The second group was created by implantation of CREF cells that were transfected with E1a and E1b of wild type adenovirus type 5 prior to transfection with T24 and pH1 (Wt tumors). Alginate beads were implanted at three different sites in the rat, i.e. subcutaneous in the flank, subcutaneous in the tail and under the renal capsule. Tumorigenicity, invasiveness and metastatic capacity of the transfectant cell lines were determined. The tumor latency period (TLP), the doubling time of the tumors and the metastatic capacity of the cell lines depended on the site of implantation. Invasion was not influenced by site-dependency. Wt tumors were invasive and generally had longer TLP than the CREF tumors. Wt tumors did not metastasize to the lungs as opposed to CREF tumors. We concluded that the genetic background of Wt cells modulated the effect of ras transfection by stretching the TLP and by limiting the metastatic potential to the draining lymph nodes. Malignancy per se was not repressed since no differences in invasive capacity were noticed.

Quantitative and qualitative differences in growth, invasion and lung colonization of an anaplastic and a papillary human thyroid cancer cell line in vitro and in vivo.

Invasion and metastasis remain major reasons for failure of anti-cancer therapy. Cell lines derived from human carcinomas are frequently used to investigate the molecular mechanisms that underlie invasion and metastasis. Unfortunately many of these cell lines do not retain the malignant characteristics of their parental tumors. We therefore conducted a series of experiments in vivo and in vitro to identify which aspects of malignancy of a papillary (NPA'87) and an anaplastic (DR090-1) thyroid carcinoma were consistent with the pathology of the parental tumor types. We evaluated tumor growth, invasion and metastasis of DRO90-1 and NPA'87 in vivo following inoculation of the tumor cells under the dermis, under the renal capsule and into the lateral tail vein of nude mice. This evaluation in vivo showed that the anaplastic carcinoma had a faster growth rate compared with the papillary carcinoma. Furthermore, the papillary carcinoma cells could destroy and infiltrate surrounding tissue but were not capable of extravasation and colonization of lung tissue. The anaplastic cells formed lung nodules following injection into the tail vein of nude mice. This lung colonizing capability of DRO90-1 correlated with their capacity to secrete an active 62 kDa gelatinase and to migrate through reconstituted basement membrane in vitro.

The novel silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin displays high lipophilicity, improved human blood stability, and potent anticancer activity.

We describe the rational design and synthesis of B- and A, B-ring-modified camptothecins. The key alpha-hydroxy-delta-lactone pharmacophore in 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (DB-67, 14) displays superior stability in human blood when compared with clinically relevant camptothecin analogues. In human blood 14 displayed a t(1/2) of 130 min and a percent lactone at equilibrium value of 30%. The tert-butyldimethylsilyl group renders the new agent 25-times more lipophilic than camptothecin, and 14 is readily incorporated, as its active lactone form, into cellular and liposomal bilayers. In addition, the dual 7-alkylsilyl and 10-hydroxy substitution in 14 enhances drug stability in the presence of human serum albumin. Thus, the net lipophilicity and the altered human serum albumin interactions together function to promote the enhanced blood stability. In vitro cytotoxicity assays using multiple different cell lines derived from eight distinct tumor types indicate that 14 is of comparable potency to camptothecin and 10-hydroxycamptothecin, as well as the FDA-approved camptothecin analogues topotecan and CPT-11. In addition, cell-free cleavage assays reveal that 14 is highly active and forms more stable top1 cleavage complexes than camptothecin or SN-38. The impressive blood stability and cytotoxicity profiles for 14 strongly suggest that it is an excellent candidate for additional in vivo pharmacological and efficacy studies.

The highly lipophilic DNA topoisomerase I inhibitor DB-67 displays elevated lactone levels in human blood and potent anticancer activity.

The novel silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB-67) is 25- to 50-times more lipophilic than camptothecin and readily incorporates into lipid bilayers. Using the method of fluorescence anisotropy titration, we determined that DB-67 bound to small unilamellar vesicles composed of dilaurylphosphatidylcholine (DLPC) with an association constant (K value) of 5000 M(-1). This association constant is significantly higher than the K(DLPC) value observed for camptothecin (K(DLPC) value of 110 M(-1)). Using HPLC methods, we demonstrated that the presence of liposomal membranes readily stabilize the lactone form of DB-67. At drug and lipid concentrations of 10 microM and 0.3 mM, respectively, the lactone form of DB-67 persisted in liposome suspension after 3 h of incubation at 37 degrees C. Thus an advantage of a liposomal formulation of DB-67 is that the presence of lipid bilayers assists with stabilizing the key pharmacophore of the agent. The highly lipophilic character of DB-67, in combination with its 10-hydroxy moiety (which functions to enhance lactone stability in the presence of human serum albumin), results in DB-67 having superior stability in human blood with a percent lactone at equilibrium value of 30 [Cancer Res. 59 (1999) 4898; J. Med. Chem. 43 (2000) 3970]. Potent cytotoxicities against a broad range of cancer cells were observed for DB-67, indicating that DB-67 is of comparable potency to camptothecin. The impressive human blood stability and cytotoxicity profiles for DB-67 indicate it is an excellent candidate for comprehensive in vivo pharmacological and efficacy studies. Based on these promising attributes, DB-67 is currently being developed under the NCI RAID program. Due to its potent anti-topoisomerase I activity and its intrinsic blood stability, DB-67 appears as an attractive novel camptothecin for clinical development.

Invasiveness in primary intracranial tumors: Part 2. Studies with scanning electron microscopy of cell surface alterations associated with invasiveness.

We previously described an experimental model for the production of invasive and noninvasive astrocytic series tumors and a semiquantitative assessment of their invasiveness. The tumors are produced by intracerebral inoculation of simian virus 40-transformed cells from four different brain regions. Cells of cerebral cortex and brain stem derivation produced invasive tumors; tumors of cerebral cortex derivation were more invasive and became increasingly invasive with alternate in vivo and in vitro passage. Cells of cerebellar hemisphere and vermis derivation produced primarily noninvasive tumors. This report describes the surface configuration of these tumor cells in culture at or near confluence as visualized with scanning electron microscopy. Normal cells form a multilayered base of flat, overlapping cells with few excrescences and indistinct borders and have only rare dividing or giant cells attached. Invasive tumor cells from a monolayer base of extremely flat and spread out cells almost devoid of excrescences, with few dividing or giant cells attached. Cells of cerebral cortex derivation additionally have numerous microvilli and ruffles at points of intercellular contact and become covered with microvilli after passage. These features were retained after cloning by dilution plating, but not after cloning by growth in soft agar. Our primarily noninvasive cells form a multilayered base of rounded cells covered with various excrescences and numerous attached dividing and giant cells. These surface features seem to be related to intercellular and cell-substrate adherence. The configuration of the invasive cells is consistent with increased cell-substrate adherence, substrate-dependent inhibition of locomotion, and decreased intercellular adherence among the more invasive cells. The configuration of our primarily noninvasive cells is consistent with decreased cell-substrate adherence and unrestricted multilayered growth. Thus, it seems that invasiveness is expressed at the cell surface and is related to disturbances in adherence balance rather than in cell proliferation.

Role of viruses in the induction of primary intracranial tumors.

The role of viruses in the induction of primary intracranial tumors is reviewed. Papovaviruses of the simian virus 40 (SV40) group are used as a representative model, and a distillation of the literature on virus induction of intracranial tumors in laboratory animals and neoplastic transformation of cells in culture is presented. The molecular sequence of events during tumor induction and neoplastic cell transformation is also discussed. Recent evidence that the papovaviruses play a role in the formation of human brain tumors is summarized.

Invasiveness in primary intracranial tumors: Part 1. An experimental model using cloned SV40 virus-produced hamster brain tumors.

This report presents an experimental model for study of the cellular and molecular biology of invasiveness in tumors. It uses SV40 virus for the production of primary intracranial tumors that are invasive for normal brain and vary markedly and predictably in this invasiveness. Cell cultures of dissociated 1- to 2-day-old Syrian hamster cerebral cortex (Cx), brain stem (Bs), cerebellar hemisphere (Cbh), and cerebellar vermis (Cbv) were transformed with SV40 virus and inoculated intracerebrally into newborn hamsters. All 368 animals that developed intracranial tumors were killed, and tumor was taken for histological and immunofluorescence studies, assessment of extent of invasiveness, and preparation of cell cultures from which cells were cloned by dilution plating or growth in soft agar. A few hamsters were perfused with glutaraldehyde for studies of tumor ultrastructure. All cloned and uncloned tumor cells were reinoculated to produce second- and third-passage tumors. Characteristic differences in morphology and growth rate were observed between normal astrocytes derived from each brain region, and these phenotypic differences were retained after virus transformation and tumor production. Cloned and uncloned Cx cell-derived tumors of second and third passage diffusely invaded adjacent normal brain, although those of first passage invaded only slightly. Except for extracerebral spread, these tumors resembled human astrocytic series tumors. Bs and some Cbh cell-derived tumors were also astrocytic but more undifferentiated and only slightly invasive; Cbv and other Cbh cell-derived tumors were sarcomatous and only extended along perivascular spaces or were not invasive at all. The tumor cells contained glial fibrillary acidic protein and SV40 T-antigen. These results suggest that astrocytes from different brain regions vary in genomic stability and support the theory that differences in invasiveness reflect the development of heterogeneity and subsequent selection of more aggressive subpopulations of cells.

Histone deacetylase inhibitors promote apoptosis and differential cell cycle arrest in anaplastic thyroid cancer cells.

Little information exists concerning the response of anaplastic thyroid carcinoma (ATC) cells to histone deacetylase inhibitors (HDAIs). In this study, the cellular response to the histone deacetylase inhibitors, sodium butyrate and trichostatin A, was analyzed in cell lines derived from primary anaplastic thyroid carcinomas. HDAIs repress the growth (proliferation) of ATC cell lines, independent of p53 status, through the induction of apoptosis and differential cell cycle arrest (arrested in G1 and G2/M). Apoptosis increases in response to drug treatment and is associated with the appearance of the cleaved form of the caspase substrate, poly-(ADP-ribose) polymerase (PARP). Cell cycle arrest is associated with the reduced expression of cyclins A and B, the increased expression of the cyclin-dependent kinase inhibitors, p21(Cip1/WAF1) and p27Kip1, the reduced phosphorylation of the retinoblastoma protein (pRb), and a reduction in cdk2 and cdk1-associated kinase activities. In ATC cells overexpressing cyclin E, drug treatment failed to replicate these events. These results suggest that growth inhibition of ATC cells by HDAIs is due to the promotion of apoptosis through the activation of the caspase cascade and the induction of cell cycle arrest via a reduction in cdk2- and cdk1-associated kinase activities.

Butyrate alters the expression and activity of cell cycle components in anaplastic thyroid carcinoma cells.

Anaplastic thyroid carcinoma (ATC) is the most malignant and aggressive form of thyroid cancer. Most patients die within months of diagnosis, primarily due to the absence of effective chemotherapeutic strategies. Identifying alternative therapies is necessary to increase long-term survival. Butyrate elicits a number of responses from cancer cells both in vitro and in vivo including growth repression, cell cycle arrest, differentiation, and apoptosis. Even though many types of cancer cells have been studied, little is known of the response of ATC cells to this drug. In this study, we report that butyrate induces differential cell cycle arrest (arrest in G1 and G2/M phases) in an ATC cell line that correlates with changes in the expression, phosphorylation, and activity of key components of the cell cycle machinery. Exposure to butyrate increases the expression of the cyclin-dependent kinase inhibitors, p21/Cip1 and p27/Kip1, decreases the expression of cyclin A and cyclin B, inhibits the phosphorylation of the retinoblastoma protein (pRb), and decreases the activity of cdk1 and cdk2-associated kinases. These results suggest that butyrate may be useful in the clinical treatment of ATC.

Translational control of malignancy: the mRNA cap-binding protein, eIF-4E, as a central regulator of tumor formation, growth, invasion and metastasis.

Recent studies have implicated the mRNA cap-binding protein, eIF-4E, as a key regulator of malignant progression. Indeed, the major intracellular signaling pathways involved in tumor growth and malignancy, the MAP kinase and PI3 kinase pathways, induce eIF-4E activity. Furthermore, immunohistochemical analyses have revealed that eIF-4E is overexpressed and related to disease progression in human cancers of the colon, head and neck, and breast. In experimental tumors, manipulation of eIF-4E function profoundly affects not only tumorigenesis but also tumor invasion and metastasis. While increasing global protein synthesis rates, the increased activity of eIF-4E that typifies both human and experimental tumors disproportionately enhances the translation of a specific array of potent growth regulatory and malignancy-related proteins, including c-myc, cyclin D1, ornithine decarboxylase, vascular endothelial growth factor, basic fibroblast growth factor and others. Herein, we review the data supporting the notion that, by coordinately upregulating the translation of numerous malignancy-related proteins, eIF-4E plays a pivotal role in regulating not only tumor growth, but also invasion and metastasis.