Targeting HER3 by interfering with its Sec61-mediated cotranslational insertion into the endoplasmic reticulum.

There is increasing evidence implicating human epidermal growth factor receptor 3 (HER3) in several types of cancer. However, the development of targeted therapies to inactivate HER3 function has been a challenging endeavor. Its kinase domain functions in allostery not catalysis, and the classical ATP-analog class of tyrosine kinase inhibitors fail to inactivate it. Here we describe a novel approach that eliminates HER3 expression. The small-molecule cotransin CT8 binds the Sec61 translocon and prevents the signal peptide of the nascent HER3 protein from initiating its cotranslational translocation, resulting in the degradation of HER3 but not the other HER proteins. CT8 treatment suppresses the induction of HER3 that accompanies lapatinib treatment of HER2-amplified cancers and synergistically enhances the apoptotic effects of lapatinib. The target selectivities of cotransins are highly dependent on their structure and the signal sequence of targeted proteins and can be narrowed through structure-function studies. Targeting Sec61-dependent processing identifies a novel strategy to eliminate HER3 function.

A tumor-suppressing function in the epithelial adhesion protein Trask.

Trask/CDCP1 is a transmembrane glycoprotein widely expressed in epithelial tissues whose functions are just beginning to be understood, but include a role as an anti-adhesive effector of Src kinases. Early studies looking at RNA transcript levels seemed to suggest overexpression in some cancers, but immunostaining studies are now providing more accurate analyses of its expression. In an immuno-histochemical survey of human cancer specimens, we find that Trask expression is retained, reduced or sometimes lost in some tumors compared with their normal epithelial tissue counterparts. A survey of human cancer cell lines also show a similar wide variation in the expression of Trask, including some cell types with the loss of Trask expression, and additional cell types that have lost the physiological detachment-induced phosphorylation of Trask. Three experimental models were established to interrogate the role of Trask in tumor progression, including two gain-of-function models with tet-inducible expression of Trask in tumor cells lacking Trask expression, and one loss-of-function model to suppress Trask expression in tumor cells with abundant Trask expression. The induction of Trask expression and phosphorylation in MCF-7 cells and in 3T3v-src cells was associated with a reduction in tumor metastases while the shRNA-induced knockdown of Trask in L3.6pl cancer cells was associated with increased tumor metastases. The results from these three models are consistent with a tumor-suppressing role for Trask. These data identify Trask as one of several potential candidates for functionally relevant tumor suppressors on the 3p21.3 region of the genome frequently lost in human cancers.

MRI methods for evaluating the effects of tyrosine kinase inhibitor administration used to enhance chemotherapy efficiency in a breast tumor xenograft model.

PURPOSE: To evaluate whether quantitative MRI parameters are sensitive to the effects of the tyrosine kinase inhibitor gefitinib and can discriminate between two different treatment protocols. MATERIALS AND METHODS: Untreated mice with BT474 breast tumor xenografts were characterized in a preliminary study. Subsequently, tumor volume, apparent diffusion coefficient (ADC), transendothelial permeability (K(ps)), and fractional plasma volume (fPV) were measured in three groups of mice receiving: 1) control vehicle for 10 days, or gefitinib as 2) a single daily dose for 10 days or 3) a 2-day pulsed dose. RESULTS: Gefitinib treatment resulted in significant tumor growth inhibition (pulsed: 439 +/- 93; daily: 404 +/- 53; control: 891 +/- 174 mm(3), P < 0.050) and lower cell density (pulsed: 0.15 +/- 0.01, daily: 0.17 +/- 0.01, control: 0.24 +/- 0.01, P < 0.050) after 9 days. Tumor ADC increased in treated groups but decreased in controls (P > 0.050). Tumor K(ps) decreased with pulsed treatment but rebounded afterwards and increased with daily treatment (P > 0.050). Tumor fPV increased in both treated groups, decreasing afterwards with pulsed treatment (P > 0.050). CONCLUSION: Quantitative MRI can provide a sensitive measure of gefitinib-induced tumor changes, potentially distinguish between treatment regimens, and may be useful for determining optimal treatment scheduling for enhancing chemotherapy delivery.

The epidermal growth factor receptor family: biology driving targeted therapeutics.

The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in regulating cell proliferation, survival, differentiation and migration. The ErbB receptors carry out both redundant and restricted functions in mammalian development and in the maintenance of tissues in the adult mammal. Loss of regulation of the ErbB receptors underlies many human diseases, most notably cancer. Our understanding of the function and complex regulation of these receptors has fueled the development of targeted therapeutic agents for human malignancies in the last 15 years. Here we review the biology of ErbB receptors, including their structure, signaling, regulation, and roles in development and disease, then briefly touch on their increasing roles as targets for cancer therapy.

Targeting HER proteins in cancer therapy and the role of the non-target HER3.

Members of the human epidermal growth factor receptor (HER) family have been of considerable interest in the cancer arena due to their potential to induce tumorigenesis when their signalling functions are deregulated. The constitutive activation of these proteins is seen in a number of different common cancer subtypes, and in particular EGFR and HER2 have become highly pursued targets for anti-cancer drug development. Clinical studies in a number of different cancers known to be driven by EGFR or HER2 show mixed results, and further mechanistic understanding of drug sensitivity and resistance is needed to realise the full potential of this treatment modality. Signalling in trans is a key feature of HER family signalling, and the activation of the PI3K/Akt pathway, so critically important in tumorigenesis, is driven predominantly through phosphorylation in trans of the kinase inactive member HER3. An increasing body of evidence shows that HER3 plays a critical role in EGFR- and HER2-driven tumours. In particular, HER3 lies upstream of a critically important tumorigenic signalling pathway with extensive ability for feedback and cross-talk signalling, and targeting approaches that fail to account for this important trans-target of EGFR and HER2 can be undermined by its resiliency and resourcefulness. Since HER3 is kinase inactive, it is not a direct target of kinase inhibitors and not presently an easily drugable target. This review presents the current evidence highlighting the role of HER3 in tumorigenesis and its role in mediating resistance to inhibitors of EGFR and HER2.

The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis.

The year 2007 marks exactly two decades since Human Epidermal Growth Factor Receptor-2 (HER2) was functionally implicated in the pathogenesis of human breast cancer. This finding established the HER2 oncogene hypothesis for the development of some human cancers. The subsequent two decades have brought about an explosion of information about the biology of HER2 and the HER family. An abundance of experimental evidence now solidly supports the HER2 oncogene hypothesis and etiologically links amplification of the HER2 gene locus with human cancer pathogenesis. The molecular mechanisms underlying HER2 tumorigenesis appear to be complex and a unified mechanistic model of HER2-induced transformation has not emerged. Numerous hypotheses implicating diverse transforming pathways have been proposed and are individually supported by experimental models and HER2 may indeed induce cell transformation through multiple mechanisms. Here I review the evidence supporting the oncogenic function of HER2, the mechanisms that are felt to mediate its oncogenic functions, and the evidence that links the experimental evidence with human cancer pathogenesis.

Targeting the function of the HER2 oncogene in human cancer therapeutics.

The year 2007 marks exactly two decades since human epidermal growth factor receptor-2 (HER2) was functionally implicated in the pathogenesis of human breast cancer (Slamon et al., 1987). This finding established the HER2 oncogene hypothesis for the development of some human cancers. An abundance of experimental evidence compiled over the past two decades now solidly supports the HER2 oncogene hypothesis. A direct consequence of this hypothesis was the promise that inhibitors of oncogenic HER2 would be highly effective treatments for HER2-driven cancers. This treatment hypothesis has led to the development and widespread use of anti-HER2 antibodies (trastuzumab) in clinical management resulting in significantly improved clinical antitumor efficacies that have transformed the clinical practice of oncology. In the shadows of this irrefutable clinical success, scientific studies have not yet been able to mechanistically validate that trastuzumab inhibits oncogenic HER2 function and it remains possible that the current clinical advances are a consequence of the oncogene hypothesis, but not a translation of it. These looming scientific uncertainties suggest that the full promise of the treatment hypothesis may not yet have been realized. The coming decade will see a second generation of HER2-targeting agents brought into clinical testing and a renewed attempt to treat HER2-driven cancers through the inactivation of HER2. Here, I review the development of treatments that target HER2 in the context of the HER2 oncogene hypothesis, and where we stand with regards to the clinical translation of the HER2 oncogene hypothesis.

The use of molecular markers in farnesyltransferase inhibitor (FTI) therapy of breast cancer.

The hypothesis that pharmacologic inhibitors of Ras can be effective anti-cancer agents has led to the development of Farnesyltransferase inhibitors (FTIs). These agents inhibit the requisite processing of a number of cellular proteins including Ras. FTIs have shown good anti-tumor efficacy and little toxicity in preclinical models and based on these results, numerous clinical trials are currently underway to evaluate the clinical potential of these agents in patients with cancer. However, contrary to the ideas that led to their design, mechanistic studies have not confirmed that they inhibit tumors through the inhibition of Ras. FTIs inhibit the growth of a broad variety of human tumor cells in vitro and studies to date have not identified cellular characteristics that predict the antitumor efficacy of this class of agents. We have studied a panel of breast cancer cell lines that differ widely in their sensitivity to FTI in order to determine which molecular characteristics may determine sensitivity to this class of agents. In these cells we find that FTI sensitivity does not correlate with the relative expression of Ras isoforms or the inhibition of Ras processing, growth factor signaling, expression of estrogen receptor or the overexpression of growth factor receptors. Looking for other molecular correlates of FTI sensitivity we have compared the activity of farnesylprotein transferase (FPTase) among these cells and although we find no overall correlation with FTI sensitivity, we find that two cell lines with unusually low FPTase activity are sensitive. Comparing p53 genotype with FTI-sensitivity we find that although most cell lines in our panel have mutant p53, all three cell lines with wild-type p53 are quite sensitive to FTI. In fact, MCF-7 cells which have both wild-type p53 and the lowest FPTase activity are the most FTI-sensitive cell type we have ever seen. Although these studies do not identify any single molecular marker that can accurately predict FTI sensitivity in breast tumors, they highlight the potential roles of FPTase activity and p53 function for further analysis.

The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells.

The epidermal growth factor receptor (EGFR) is commonly overexpressed in many human tumors and provides a new target for anticancer drug development. ZD1839 ("Iressa"), a quinazoline tyrosine kinase inhibitor selective for the EGFR, has shown good activity in preclinical studies and in the early phase of clinical trials. However, because it remains unclear which tumor types are the best targets for treatment with this agent, the molecular characteristics that correlate with tumor sensitivity to ZD1839 have been studied. In a panel of human breast cancer and other epithelial tumor cell lines, HER2-overexpressing tumors were particularly sensitive to ZD1839. Growth inhibition of these tumor cell lines was associated with the dephosphorylation of EGFR, HER2, and HER3, accompanied by the loss of association of HER3 with phosphatidylinositol 3-kinase, and down-regulation of Akt activity. These studies suggest that HER2-overexpressing tumors are particularly susceptible to the inhibition of HER family tyrosine kinase signaling and suggest novel strategies to treat these particularly aggressive tumors.

Oral gossypol in the treatment of patients with refractory metastatic breast cancer: a phase I/II clinical trial.

Gossypol has demonstrated in vitro effects on cell cycle regulation and anti-tumor activity against mammary carcinoma cell lines. This Phase I/II study assesses both the effect of gossypol on cell cycle regulatory proteins in vivo and the clinical effect. Twenty women with refractory metastatic breast cancer received oral gossypol at daily doses between 30 and 50 mg per day. Gossypol plasma levels were measured (n = 8) and the modulation of the retinoblastoma (Rb) gene protein and Cyclin D1 was assessed by serial biopsies (n = 4). Grade I-II toxicities with gossypol treatment included nausea in 30% of patients, fatigue 15%, emesis 15%, altered taste sensation 15% and diarrhea in 10% of patients. Two of the three patients receiving 50 mg/day experienced dose limiting dermatologic toxicity (grade III). One patient had a minor response and two patients had stable disease with > 50% decline in serial assessments of the serum tumor markers. Immunohistochemical analysis of cyclin D1 and Rb expression in serial biopsies of four patients revealed both a concurrent decrease in cyclin D1 expression and an increase in nuclear Rb expression in three patients. The maximal tolerated dose (MTD) of gossypol was 40 mg/day. Gossypol appears to affect the expression of Rb protein and cyclin D1 in breast cancer metastases at doses achievable, yet had negligible antitumor activity against anthracycline and taxane refractory metastatic breast cancer. The cell cycle regulatory effects of gossypol suggest a potential role for gossypol as a modulating agent in conjunction with other cell cycle specific compounds.

Inhibition of heat shock protein 90 function by ansamycins causes the morphological and functional differentiation of breast cancer cells.

17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) is an ansamycin antibiotic that binds to a conserved pocket in Hsp90 and induces the degradation of proteins that require this chaperone for conformational maturation. 17-AAG causes a retinoblastoma (RB)-dependent G1 block in cancer cells and is now in clinical trial. In breast cancer cells, G1 block is accompanied by differentiation and followed by apoptosis. The differentiation is characterized by specific changes in morphology and induction of milk fat proteins and lipid droplets. In cells lacking RB, neither G1 arrest nor differentiation occurs; instead, they undergo apoptosis in mitosis. Introduction of RB into these cells restores the differentiation response to 17-AAG. Inhibitors of the ras, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways cause accumulation of milk fat proteins and induction of lipid droplets when associated with G1 arrest but do not cause morphological changes. Thus, regulation of Hsp90 function by 17-AAG in breast cancer cells induces RB-dependent morphological and functional mammary differentiation. G1 arrest is sufficient for some but not all aspects of the phenotype. Induction of differentiation may be responsible for some of the antitumor effects of this drug.

Farnesyl:protein transferase inhibitors as potential agents for the management of human prostate cancer.

The effects of farnesyl:protein transferase inhibitors (FTIs) were evaluated against hormone-dependent and hormone-independent prostate cancer cell lines harboring mutant and wild type Ras. The combinations of the FTI with hormones and chemotherapy were explored. The effect of FTI on the growth of human prostate cancer lines was examined under anchorage-dependent and -independent conditions. Changes in Ras processing and cellular localization were examined by immunoblotting and immunocytochemistry. Hormone-dependent (LNCaP) and -independent (TSU-Pr1, PC3 and DU145) human prostate cancer cell lines were growth-inhibited by the FTI L-744,832 at concentrations ranging from 100 nM to 20 &mgr;M. The inhibition was accompanied by loss of protein farnesylation and with the accumulation of Ha-Ras as its unprocessed, cytosolic form. No effect on N- and Ki-Ras processing was observed. The transformed phenotype of TSU-Pr1 cells, which possess a Ha-Ras Gly-12-Val activating mutation, reverted following FTI treatment. Enhanced antitumor effects were observed when the FTI was combined with gamma-radiation, etoposide, doxorubicin, cisplatin, estramustine and the antihormone bicalutamide. In particular, the combination of taxol and FTI was synergistic for DU145 cells, a cell line that is only marginally sensitive to the FTI alone. The sensitivity of human prostate cancer cell lines to the FTI is independent of the presence of mutations of tumor suppressors, cell cycle regulators and of the activation of a variety of oncogenes, including Ras. A cell line expressing mutated Ha-Ras is particularly sensitive. Enhanced antitumor effects were observed with an anti-androgen, gamma-irradiation, and several chemotherapeutic agents. These findings support the clinical evaluation of FTIs alone or in combination as treatment for this disease. Prostate Cancer and Prostatic Diseases (2001) 4, 33-43

Inhibition of Src kinases by a selective tyrosine kinase inhibitor causes mitotic arrest.

src kinase activity is elevated in some human tumors, including breast and colon cancers. The precise cellular function of the src family kinases is not clearly understood, but they appear to be involved in numerous signaling pathways. We studied the effects of PD173955, a novel src family-selective tyrosine kinase inhibitor, on cancer cell lines and found that it has significant antiproliferative activity due to a potent arrest of mitotic progression. The mitotic block occurs after chromosome condensation in prophase, before spindle assembly and without loss of cyclin A and B kinase activities. This effect is seen in cancer cell lines of all types with low or high activities of src kinases as well as in untransformed cell lines. In MDA-MB-468 breast cancer cells, this drug produces a rapid inhibition of cellular src and yes kinase activities as well as suppression of the mitotic hyperactivity of these kinases. This compound defines a novel class of antimitotic drugs that work through inhibition of src kinases and possibly other protein kinases that are required for progression through the initial phases of mitosis.

Farnesyl transferase inhibitors cause enhanced mitotic sensitivity to taxol and epothilones.

An important class of cellular proteins, which includes members of the p21ras family, undergoes posttranslational farnesylation, a modification required for their partition to membranes. Specific farnesyl transferase inhibitors (FTIs) have been developed that selectively inhibit the processing of these proteins. FTIs have been shown to be potent inhibitors of tumor cell growth in cell culture and in murine models and at doses that cause little toxicity to the animal. These data suggest that these drugs might be useful therapeutic agents. We now report that, when FTI is combined with some cytotoxic antineoplastic drugs, the effects on tumor cells are additive. No interference is noted. Furthermore, FTI and agents that prevent microtubule depolymerization, such as taxol or epothilones, act synergistically to inhibit cell growth. FTI causes increased sensitivity to induction of metaphase block by these agents, suggesting that a farnesylated protein may regulate the mitotic check point. The findings imply that FTI may be a useful agent for the treatment of tumors with wild-type ras that are sensitive to taxanes.

Derivation and characterization of retinoid-resistant human embryonal carcinoma cells.

The retinoids exert potent growth and differentiation effects on normal and neoplastic cells through two families of nuclear receptors. These are the retinoic acid receptors (RAR alpha, RAR beta, RAR gamma) and the retinoid-X receptors (RXR alpha, RXR beta, RXR gamma). All-trans retinoic acid (RA) induces terminal neuronal differentiation and represses tumorigenicity of the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1). Hexamethylene bisacetamide (HMBA) induces a phenotype distinct from RA-treated NT2/D1 cells. This study reports the derivation and characterization of RA- and HMBA-resistant NT2/D1 clones. Nine RA-resistant (NT2/D1-R1 through NT2/D1-R9) and one HMBA-resistant (NT2/D1-H1) clones were derived after mutagen treatment of NT2/D1 cells and selection in RA or HMBA. NT2/D1-R cells were cross-resistant to 9-cis retinoic acid (9-cis RA), a ligand activating the RAR and RXR pathways, but retained maturation response to HMBA. A representative RA-resistant clone, NT2/D1-R1, overcame the antitumorigenic actions of RA as assessed in athymic mice. NT2/D1-H1 cells were dually resistant to RA and 9-cis RA. All these retinoid resistant cells exhibit deregulated expression of RAR gamma but not RAR alpha or RAR beta. Southern analysis using RAR gamma probes shows no apparent structural differences in genomic DNA between NT2/D1 cells and the RA-resistant subclones. Pulsed-field gel electrophoresis (PFGE) with RAR gamma probes demonstrated an Mlu-I restriction fragment length polymorphism, but no other structural abnormalities in these cells or a panel of germ cell tumor (GCT) cell lines. Full-length RAR gamma 1 coding region cDNAs were cloned from NT2/D1 and NT2/D1-R1 cells and these sequences were identical, suggesting RA resistance in these cells is due to altered regulation of RAR gamma. These differentiation-resistant cells are useful to study RAR gamma target genes or mechanisms engaged by these differentiation inducing agents in human embryonal carcinomas.

Growth suppression of transformed human bronchial epithelial cells by all-trans-retinoic acid occurs through specific retinoid receptors.

The retinoids are reported to chemoprevent second aerodigestive tract cancers in patients with prior lung or head and neck cancers. Since those retinoids already examined in clinical trials do not induce major clinical responses in lung cancers, it is hypothesized that the beneficial chemoprevention activity in lung neoplasias occurs within 'fields' of carcinogen-transformed epithelial cells. To begin to investigate this retinoid action during lung carcinogenesis, the BZR-T33 ras transformed human bronchial epithelial cell line that grows in an anchorage independent manner was examined. This study reports, as compared to controls, that all-trans-retinoic acid (RA)-treatment suppresses BZR-T33 proliferation in monolayer cultures and in anchorage independent and cloning efficiency growth assays. RA induces RAR-gamma 2 > RAR gamma 1 in BZR-T33 cells but expression at the total cellular RNA level of RAR alpha and RXR alpha is not augmented by RA-treatment. RAR beta mRNA expression is repressed before and after RA-treatment and is only detected using a reverse transcription polymerase chain reaction (RT-PCR) assay. To determine directly which of these expressed retinoid receptors signals growth suppression, each receptor was individually transfected into BZR-T33 cells using episomal vector in colony efficiency assays. RAR gamma over-expression in the presence or absence of RA-treatment did not suppress BZR-T33 growth more than controls. In contrast, over-expression of the other examined retinoid receptors inhibited BZR-T33 cellular cloning efficiency prior to RA-treatment and in this decreasing order after RA-treatment: RAR alpha > RAR beta > RXR alpha. The findings reported here reveal that RA suppresses proliferation and cloning efficiency in this transformed human bronchial epithelial cell through specific retinoid receptors. Further work is needed to evaluate the role of RA or its nuclear receptors in inhibiting even earlier steps in lung carcinogenesis.

all-trans retinoic acid for treating germ cell tumors. In vitro activity and results of a phase II trial.

BACKGROUND: Germ cell tumors (GCTs) are characterized by a capacity to differentiate in vivo and in vitro. The authors' previous work highlighted the finding that retinoid-mediated GCT differentiation in vitro correlates with expression of the retinoic acid receptor-gamma. METHODS: Six human GCT cell lines were studied to determine whether their growth and differentiation were linked to the retinoic acid response pathway. The maturation and growth inhibitory responses to all-trans retinoic acid (RA) were studied as well as the expression of retinoic acid receptor-gamma (RAR-gamma). The clinical antitumor activity of RA was studied in a Phase II trial of RA in patients with chemotherapy-refractory GCTs. RESULTS: The RA response pathway was correlated with the control of growth and maturation in three of the six GCT cell lines studied. In one GCT cell line, RA induced RAR-gamma expression and terminal differentiation. In a second, there was high basal expression of RAR-gamma and poor basal proliferative potential. A third cell line showed a more mature basal phenotype than other cell lines studied and marked growth inhibition when treated with RA. Sixteen patients were treated with RA for an average of 7 weeks in the clinical trial. No complete or partial responses were noted. CONCLUSION: A Phase II clinical trial of RA failed to show significant clinical antitumor activity in patients with chemotherapy-refractory GCTs. However in vitro data suggest that the control of growth and maturation in some GCT cell lines involve the RA signaling pathway. Further studies are warranted to define the role that other retinoids with receptor selectivity or more favorable pharmacologic properties may play in the maturation or antitumor responses of GCTs.

Growth suppression of acute promyelocytic leukemia cells having increased expression of the non-rearranged alleles: RAR alpha or PML.

The balanced t(15;17) rearrangement found in acute promyelocytic leukemia (APL) cells fuses PML on chromosome 15 to the retinoic acid receptor alpha (RAR alpha) on chromosome 17. PML/RAR alpha is expressed in APL cells with the non-rearranged alleles, PML and RAR alpha. Clinical remissions induced by all-trans-retinoic acid (RA) treatment of APL patients are linked to expression of PML/RAR apha, a transcription factor with reported dominant negative functions. The roles of PML and RAR alpha in the RA response of APL have not yet been fully explored. This study examines these roles by individually transfecting RAR alpha and PML into NB4 APL cells. NB4 is the sole APL cell line containing the t(15;17). RA treatment represses NB4 cell growth and induces a myeloid phentoype. Full length cDNAs for RAR alpha and PML were individually cloned into a CMV-driven expression vector containing the neomycin resistance gene. Surprisingly, none of the obtained stable transfectants expressed exogenous RAR alpha or PML mRNAs even when reverse transcription polymerase chain reaction (RT-PCR) detection assays were used. All clones expressed the neomycin resistance gene and were similar to parental NB4 cells in their growth and differentiation properties. An explanation explored for this lack of gene expression was that increased levels of RAR alpha or PML might suppress APL cell growth. To examine this possibility, transfection experiments were repeated using an episomal vector-based expression system containing an SV40 driven RAR alpha or PML cDNA and the hygromycin B resistance gene. A new selection strategy augmented expression of the desired cDNAs. A control episomal vector lacked a cDNA insert. Following electroporation and selection, exogenous RAR alpha expression was obtained. Compared to controls, the growth of these transfectants was markedly inhibited before and after RA-treatment and these cells more prominently induced myeloid maturation markers. In contrast, exogenous PML expression was transient since these transfectants did not appear to propagate in culture. These findings indicate: (1) a growth disadvantage for NB4 cells having increased expression of RAR alpha or PML and (2) increased RAR alpha expression augmented RA-mediated maturation of NB4 cells. This implicates a role for RAR alpha or PML in regulating the growth or differentiation of APL cells. It is hypothesized this occurs through antagonism of PML/RAR alpha actions in these leukemic cells.

Overexpression of the retinoic acid receptor gamma directly induces terminal differentiation of human embryonal carcinoma cells.

All-trans retinoic acid (RA) exerts profound effects on the growth and differentiation of normal, embryonic, and malignant cells. The effects of RA are mediated through multiple members of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families of nuclear transcription factors. The RARs and RXRs exhibit specific patterns of expression during development and in adult tissues suggesting tissue or cell-type specific functions. Using NTera2/clone D1 (NT2/D1) human embryonal carcinoma cells as a model, we report that the RA induced terminal differentiation of these cells into a neuronal phenotype is characterized by an increase in expression of RAR alpha, RAR beta, RAR gamma, and a slight induction of RXR alpha. To study the role of these receptors in the differentiation process we individually overexpressed RAR alpha, beta, gamma and RXR alpha in NT2/D1 cells by cDNA transfection. Using induced cDNA expression by episomal vector amplification we show that RAR gamma over-expression causes the terminal mesenchymal differentiation of these cells while over-expression of RAR alpha, beta and RXR alpha has no observed maturation or growth inhibitory effects. Over-expression of these receptors in the derived RA resistant subclone NT2/D1-R1 showed phenotypic changes characteristic of RA response in RAR gamma transfectants. These studies indicate that of the retinoid receptors expressed in RA-treated NT2/D1 cells, it is the upregulation of RAR gamma that specifically induces the terminal differentiation of these cells.

Response and resistance to retinoic acid are mediated through the retinoic acid nuclear receptor gamma in human teratocarcinomas.

All-trans-retinoic acid (RA) treatment of the multipotent human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype. Compared to untreated NT2/D1 cells, RA treated cells have reduced proliferative potential. To identify which retinoic acid receptor (RAR) is directly linked to RA response in these cells, nine RA resistant subclones were derived and characterized. Unlike parental NT2/D1 cells, all these subclones and a de novo RA resistant human TC cell line, N2102Ep clone 2AG (abbreviated N2102ep), exhibited reduced RAR gamma expression. The RAR gamma gene was studied within NT2/D1 cells and a representative RA resistant NT2/D1 subclone called NT2/D1-R1 by Southern analysis and by the transcriptional properties of cloned RAR gamma cDNAs. No structural or functional differences between these RAR gamma species were found suggesting that RA resistance is due to reduced levels of RAR gamma expressed in NT2/D1-R1 cells. To explore this possibility an RAR gamma cDNA was stably transfected into NT2/D1-R1 cells. Expression of this cDNA partially restored the response to RA in NT2/D1-R1 cells. The role of RAR gamma in parental NT2/D1 cells was studied in transient transfection assays using an FGF4 promoter-enhancer reporter construct that is transcriptionally active in undifferentiated but not in differentiated TC cells. The dose dependent co-transfection of an expressed RAR gamma cDNA with this reporter more efficiently repressed its transcriptional activity in the presence of RA than transfection of the reporter without expressed RAR gamma. Co-transfection also decreased reporter activity in the absence of exogenously added ligand. Together, these findings reveal that RAR gamma expression is tightly coupled to RA response and resistance in human TC cells. These data implicate an important role for RAR gamma in the RA-mediated differentiation of these TCs.