Classical and novel retinoids: their targets in cancer therapy.

Retinoids are important mediators of cellular growth and differentiation. Retinoids modulate the growth of both normal and malignant cells through their binding to retinoid nuclear receptors and their subsequent activation. While retinoids have demonstrated therapeutic efficacy in the treatment of acute promyelocytic leukemia, their spectrum of activity remains limited. Other agents such as histone deacetylase inhibitors may significantly increase retinoid activity in a number of malignant cell types. The novel retinoids N-(4-hydroxyphenyl) retinamide (4-HPR) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN) induce apoptosis in a wide variety of malignant cells. Their mechanism(s) of action remain unclear, although a number of potential targets have been identified. Whether the retinoid receptors are involved in 4-HPR and CD473/AHPN mediated apoptosis remains unclear. Both 4-HPR and CD437/AHPN display significant potential as therapeutic agents in the treatment of a number of premalignant and malignant conditions.

Cloning of a novel EGFR-related peptide: a putative negative regulator of EGFR.

Although epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, differentiation, and transformation in many tissues, little is known about the factor(s) that may modulate its function. We have isolated a cDNA clone from the rat gastroduodenal mucosa whose full length revealed 1,958 bp that contained 227 bp of 5'-untranslated region (UTR) and an open-reading frame encoding 479 amino acids, followed by 290 bp of 3'-UTR. It showed ~85% nucleotide homology to the external domain of the rat EGFR. We refer to the product of the newly isolated cDNA as EGFR-related protein (ERRP). In Northern blot analysis with poly(A)(+) RNA from different rat tissues, ERRP cDNA hybridized to several mRNA transcripts with the strongest reaction noted with a transcript of approximately 2 kb. Maximal expression of the 2-kb mRNA transcript was observed in the small intestine, followed by colon, liver, gastric mucosa, and other tissues. Transfection of ERRP cDNA into a colon cancer cell line, HCT116, resulted in a marked reduction in proliferation in monolayer and colony formation in soft agar compared with the vector-transfected controls. In another colon cancer cell line, Caco-2, with a tetracycline-regulated promoter system, induction of ERRP expression in the absence of doxycycline was associated with a marked reduction in EGFR activation and proliferation. We conclude that the ERRP cDNA may represent a new member of the EGFR gene family and that ERRP plays a role in regulating cell proliferation by modulating the function of EGFR.

Okadaic acid-mediated induction of the c-fos gene in estrogen receptor-negative human breast carcinoma cells utilized, in part, posttranscriptional mechanisms involving adenosine-uridine-rich elements.

Signal transduction via modulation of phosphorylation after selective inhibition of protein phosphatase (PP) 1 and/or PP2A appears to play a role in okadaic acid (OA)-mediated effects. Treatment of several estrogen receptor-negative human breast carcinoma (HBC) cells with 100 nM OA resulted in induction of c-fos, c-myc, and cyclin-dependent kinase inhibitor p21WAF1/CIP1 genes. Transfections of various luciferase reporter constructs in HBC cells revealed involvement of activator protein-1-dependent as well as -independent pathways in induction of the c-fos gene by OA. MDA-MB-468 HBC cells were stably transfected with plasmids expressing luciferase, chimeric luciferase- c-fos 3' untranslated region (3'UTR), or chimeric luciferase-p21WAF1/CIP 3'UTR mRNAs. Expression of chimeric luciferase-c-fos and luciferase-p21WAF1/CIP1 mRNAs was elevated by OA in several independent sublines. Actinomycin D chase experiments revealed an enhanced rate of decay of luciferase-c-fos mRNA, whereas treatment with OA caused approximately 3.5-fold enhanced stability of the chimeric luciferase-c-fos mRNA only. By transfecting different plasmids containing deletions of c-fos 3'UTR, OA-responsive sequences were mapped to an 86-nucleotide, AU-rich region. UV cross-linking experiments using HBC cell cytosolic proteins showed multiple complexes with the AU-rich region subfragments of c-fos, as well as c-myc and p21WAF1/CIP1 mRNAs. OA enhanced binding of a novel Mr approximately 75,000 protein present in the cytosolic extracts of HBC cells to the AU-rich RNA probes of all of the above three genes. Taken together, OA regulation of HBC cell gene expression involves the activator protein-1 pathway, as well as enhanced binding of a novel Mr approximately 75,000 protein to an AU-rich region of the 3'UTRs of the target genes.

Identification of a unique binding protein specific for a novel retinoid inducing cellular apoptosis.

The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN, CD437) induces apoptosis in a variety of cell types, many of which are cancer cells that resist the antiproliferative and/or differentiating effects of retinoids. While the retinoids exert their effects by binding to the retinoic acid nuclear receptors (RARs) or retinoid X receptors (RXRs), AHPN (CD437) binds to another protein with different ligand specificity. In nuclear extracts from HL-60R cells the binding of AHPN (CD437) was only minimally competed by either retinoic acid (tRA)or 9-cis-retinoic acid (9-cis-RA), the natural ligands for the RARs and RXRs, respectively. Moreover, AHPN (CD437) was unable to compete with either tRA or 9-cis-RA for binding to endogenous retinoid receptors in nuclear extracts from the MDA-MB-468 breast carcinoma cell line. Size exclusion chromatography revealed AHPN binding to a 95 kDa protein(s) which is neither an RAR or RXR. Our results suggest that apoptosis induction by AHPN (CD437) may occur through interaction with another protein and is independent of the RAR/RXR-signaling pathways.

S-phase arrest and apoptosis induced in normal mammary epithelial cells by a novel retinoid.

The addition of all-trans-retinoic acid has been found to mediate a G1 cell cycle phase arrest but not apoptosis in normal mammary epithelial cells. We have now found that addition of the novel retinoid 6-[3-(1-adamantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which appears to function through a pathway independent of retinoic acid nuclear receptors, results in an S-phase arrest that is preceded by a 4-fold elevation in the levels of the cyclin-cyclin dependent kinase (cdk) inhibitor p21WAF1/CIP1. Failure to inhibit E2F-1 activation of genes through its phosphorylation by the cyclin cdk2 kinase has been shown to result in S-phase arrest and apoptosis in a number of cell types. Although exposure of the normal mammary cells to CD437 does not result in modulation of cyclin A or cdk2 levels, an increase in E2F-1 levels and a marked inhibition of cyclin A/cdk2 kinase activity are observed. Exposure to CD437 results in enhanced E2F-1 binding to its DNA consensus sequences and transcriptional activity during S phase. We hypothesize that this enhanced E2F-1 transcriptional activity results in S-phase arrest and subsequent apoptosis that has been observed in other systems.

Interaction of the PA2G4 (EBP1) protein with ErbB-3 and regulation of this binding by heregulin.

The processes by which ErbB-3, an inactive tyrosine kinase, exerts its biological effects are poorly understood. Using the yeast two-hybrid system, we have isolated an ErbB-3 binding protein (Ebp1) that interacts with the juxtamembrane domain of ErbB-3. This protein is identical to that predicted to be encoded for by the human PA2G4 gene. Ebp1 is the human homologue of a previously identified cell cycle-regulated mouse protein p38-2G4. Two transcripts of ebp1 mRNA (1.7 and 2.2 kb) were detected in several normal human organs. The interaction of Ebp1 with ErbB-3 was examined in vitro and in vivo. The first 15 amino acids of the juxtamembrane domain of ErbB-3 were essential for Ebp1 binding in vitro. Treatment of AU565 cells with the ErbB-3 ligand heregulin resulted in dissociation of Ebp1 from ErbB-3. Ebp1 translocated from the cytoplasm into the nucleus following heregulin stimulation. These findings suggest that Ebp1 may be a downstream member of an ErbB-3-regulated signal transduction pathway.

Post-transcriptional regulation of the DNA damage-inducible gadd45 gene in human breast carcinoma cells exposed to a novel retinoid CD437.

The biologically active synthetic retinoid CD437 (6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene, AHPN) and different human breast carcinoma (HBC) cell lines were used to examine the possible mechanism(s) of gadd45 induction. Northern blot analysis of mRNA isolated from MCF-7, MDA-MB-468 and MDA-MB-231 HBC cell lines demonstrated a progressive increase in the 1.4 kb gadd45 transcript after exposure to 1 microM CD437. Western blot analysis showed increased gadd45 protein levels in MDA-MB-468 HBC cells following exposure to CD437. CD437 increased gadd45 mRNA levels by approximately 20-fold in MDA-MB-468 cells, however, the transcriptional activity was increased approximately 2-3-fold as demonstrated by the human gadd45 promoter-luciferase reporter construct and nuclear run-off assays. Sublines of MDA-MB-468 HBC cells expressing stably integrated GADD45 cDNA fragments were obtained and CD437-dependent induction of GADD45 analyzed. We report that approximately 300 nt located in the 5"-untranslated region (5"-UTR) of gadd45 mRNA are involved in the CD437-dependent 4-fold enhanced stability of gadd45 transcripts. MDA-MB-468 cells were stably transfected with either a plasmid having a CMV promoter-driven rabbit beta-globin gene or plasmids having a CMV promoter-driven chimeric gadd45 5"-UTR-rabbit beta-globin gene, where the entire gadd45 5"-UTR (from +1 to +298) or a 45 bp subfragment of the gadd45 5"-UTR (from +10 to +55) was positioned at the 5"-end of the rabbit beta-globin gene. CD437 was found to up-regulate expression of both the chimeric gadd45 -rabbit beta-globin transcripts, suggesting that cis element(s) involved in the CD437-dependent enhanced stability of gadd45 mRNA are contained in the 45 nt of the 5"-UTR of the gadd45 mRNA.

Activation of the p38 and JNK/SAPK mitogen-activated protein kinase pathways during apoptosis is mediated by a novel retinoid.

6-[3-(1-Adamantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a novel retinoid which induces apoptosis in the retinoic acid-resistant HL-60R human leukemia cell line. CD437-mediated poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis of HL-60R cells does not require gene transcription or protein synthesis since it occurs in the presence or absence of either actinomycin D or cycloheximide. Marked activation of both the p38 and the JNK/SAPK serine and threonine kinases occurs at 1 h of exposure to CD437 with subsequent PARP cleavage at 2 h and apoptosis noted at 4 to 6 h. CD437 concentrations as little as 10 nM result in p38 activation and apoptosis of HL-60R cells. However, inhibition of p38 activation utilizing the specific inhibitor SB203580 does not block CD437-mediated PARP cleavage or apoptosis. In addition, p38 activation is dependent upon the activation of the caspase system since p38 activation is blocked by the pan ICE inhibitor Z-VAD fmk, which also inhibits CD437-mediated apoptosis and PARP cleavage in these cells. CD437-mediated activation of JNK/SAPK is not inhibited by Z-VAD fmk, suggesting that it lies upstream of CD437 activation of caspase activity and subsequent apoptosis. The role of JNK/SAPK activation in CD437-mediated apoptosis remains to be defined.

A multidrug resistance transporter from human MCF-7 breast cancer cells.

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.

Overexpression of bcl-2 or bcl-XL fails to inhibit apoptosis mediated by a novel retinoid.

Overexpression of bcl-2 or bcl-XL has been found to inhibit the induction of apoptosis in malignant cells by a large number of agents including a wide variety of chemotherapeutic drugs. CD437 ¿6-[3-(1-adamantyl)-4 hydroxyphenyl]-2-naphthalene carboxylic acid¿ is a novel retinoid that induces apoptosis in a number of malignant cells through a unique mechanism of action. The addition of 1 microM CD437 to HL-60/NEO cells resulted in capase 3 (CPP32) activation and poly(ADP-ribose) polymerase (PARP) cleavage in 3 h whereas in bcl-2- or bcl-XL-overexpressing HL-60 cells CD437 induced CPP32 activation and PARP cleavage in 6 h. Although 50 and 300 nM CD437 were required to induce PARP cleavage in HL-60/NEO and HL-60/bcl-2, HL-60/bcl-XL cells, respectively, maximal apoptosis in both cell lines was achieved utilizing 300 nM CD437. All three cell lines, however, share identical dose-response curves in terms of their growth inhibition, suggesting that CD437-mediated inhibition of growth and induction of apoptosis represent two distinct and separable processes. In addition, CD437 induces GI arrest as well as p21WAFI/CIPI mRNA expression in these cells despite the overexpression of bcl-2 or bcl-XL. CD437 induced mitochondrial instability as indicated by cytochrome c leakage into the cytoplasm in all three cell lines. CD437 also induced growth inhibition and apoptosis of an apoptosis-resistant variant of the HL-60 cell line (HCW-2), which switched expression from bcl-2 to bcl-XL. CD437-mediated apoptosis is not accompanied by downregulation of bcl-2 or bcl-XL or upregulation of bax. The reason for the inability of bcl-2 or bcl-XL overexpression to inhibit CD437-mediated apoptosis is unclear. The ability of CD437 to initiate apoptosis in a spectrum of malignant cells without interference from bcl-2 or bcl-XL overexpression suggests that CD437 may possess significant therapeutic potential in the treatment of malignancy.

Gene amplification and transcriptional upregulation of the sarco/endoplasmic reticulum Ca2+ transport ATPase in thapsigargin-resistant hamster smooth muscle cells.

We have selected a series of cell lines from the parental Syrian hamster smooth muscle cell line DDT1-MF2that are resistant to thapsigargin (TG), a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+transport ATPases (SERCAs). Cells were selected for resistance to TG in the presence or absence of cyclosporin (CSA), which is a competitive inhibitor of the multidrug transporter p-glycoprotein (pgp). Since TG is a known substrate for pgp, selection for TG resistance was carried out in the presence of CSA in an attempt to minimize the contribution of pgp, and to identify the potential range of adaptive responses of the SERCA pump itself, during the development of the TG-resistant phenotype. Irrespective of whether the selection is carried out in the presence or absence of CSA, pgp is overexpressed in the TG-resistant DDT1-MF2cells. SERCA protein is also overproduced in the TG-resistant cell lines, which occurs through one of several mechanisms. Included among these, is amplification of the SERCA gene and enhanced transcription of the gene. Enhanced transcription is observed only upon long-term selection and occurs through the SERCA gene proximal promoter elements. Although SERCA transcription in wild-type cells is dependent upon the -284 to -72 bp region of the SERCA promoter, the TG-resistant cells utilize both the -284 to -72 bp and the -72 to +80 bp promoter regions for enhanced SERCA transcription. That is, additional elements within the -72 to +80 bp region are recruited in the TG-resistant cells to allow for increased SERCA expression. A post-transcriptional step may also be recruited by the TG-resistant cells in their overall strategy to produce increased amounts of the SERCA protein. These studies demonstrate that the DDT1-MF2cells can utilize different mechanisms which lead to increased levels of SERCA protein as the cells adapt to inhibition of the ATPase by TG.

Specific substitutions at amino acid 256 of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase mediate resistance to thapsigargin in thapsigargin-resistant hamster cells.

High levels of resistance to thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCAs), can be developed in culture by stepwise exposure of mammalian cells to increasing concentrations of TG. We have identified, in two independently selected TG-resistant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/TG, a T --> C change at nucleotide 766 introduces a Phe256 --> Leu alteration within the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4 microM, a T --> C change at nucleotide 767 introduces a Phe256 --> Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2+ transport function and ATP hydrolytic activity are not altered by such mutations. However, a 4-5-fold resistance to TG inhibition of Ca2+ transport function occurs upon the introduction of either the Phe256 --> Leu or the Phe256 --> Ser mutation into wild-type SERCA1. These specific mutations also render the hydrolytic activity of the ATPase resistant to inhibition by TG. Our results not only implicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.

Transcriptional repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 gene mediated by cis elements present in the 3'-untranslated region.

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 plays a major role in the induction of G1 cell cycle arrest following DNA damage and is known to be regulated by p53-dependent and -independent pathways. Here, we show that p21WAF1/CIP1 transcription is also regulated, independently of p53, by the cis elements that are located downstream of the transcription start site. A cDNA fragment of approximately 180 bp, located 260 bases 3' to the translation termination codon of p21WAF1/CIP1 cDNA, was cloned in both the sense and antisense orientations downstream of the CMV promoter, upstream of the SV40 promoter, and both upstream and downstream of the p21WAF1/CIP1 promoter in the plasmids carrying the luciferase reporter gene. The constructs were transiently transfected in human breast carcinoma cells MCF-7 and MDA-MB-468 and a Syrian hamster smooth muscle cell line DDT1MF2 and were found to elicit 2-3-fold or higher repression of luciferase activities. By using overlapping deletions of the above 180-bp fragment, we identified a 48-bp subfragment that contains putative cis element(s) that participate in the transcriptional repression of the p21WAF1/CIP1 gene. The overlapping subfragments bind, in vitro, to specific proteins present in the nuclear extracts of MDA-MB-468 and DDT1MF2 cells. We, therefore, propose that additional mechanism(s) exist that regulate expression of the cellular p21WAF1/CIP1 and may contribute to p21WAF1/CIP1-dependent control of the cell cycle.

TGT3, thyroid transcription factor I, and Sp1 elements regulate transcriptional activity of the 1.3-kilobase pair promoter of T1alpha, a lung alveolar type I cell gene.

Alveolar type I epithelial cells form the major surface for gas exchange in the lung. To explore how type I cells differ in gene expression from their progenitor alveolar type II cells, we analyzed transcriptional regulation of T1alpha, a gene expressed by adult type I but not type II cells. In vivo developmental patterns of T1alpha expression in lung and brain suggest active gene regulation. We cloned and sequenced 1.25 kilobase pairs of the T1alpha promoter that can drive reporter expression in lung epithelial cell lines. Deletion analyses identified regions important for lung cell expression. The base pair (bp) -100 to -170 fragment conferred differential regulation in lung epithelial cells compared with fibroblasts. Sequence alignment of this fragment with type II-specific surfactant protein B and C promoters shows similar consensus elements arranged in a different order. Gel retardation studies with alveolar epithelial cell line nuclear extracts, thyroid transcription factor I (TTF-1) homeodomain, hepatic nuclear factor (HNF)-3beta, or Sp1 proteins, and supershift assays were used to characterize TTF-1, HNF-3 (TGT3), and Sp1/Sp3 binding sites. The TGT3 site binds factors with binding properties similar to HNF-3/Fkh (hepatic nuclear factor-3/forkhead) proteins but different from HNF-3alpha or HNF-3beta. Co-transfection with a TTF-1 expression vector moderately transactivated the -170 bp-reporter construct. Mutational analysis of these three binding sites showed reduced transcriptional activity of the -170 bp promoter. Therefore, several regulatory sequences involved in type II cell gene regulation are also present in the T1alpha promoter, suggesting that genes of the peripheral lung epithelium may be regulated by similar factors.

Retinoid induced apoptosis in leukemia cells through a retinoic acid nuclear receptor-independent pathway.

Trans retinoic acid (RA) has proven to be a potent therapeutic agent in the treatment of acute promyelocytic leukemia. Unfortunately, other subtypes of acute myelogenous leukemia are resistant to the antiproliferative and differentiating effects of RA. In this report, we describe a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN; CD437) that not only totally inhibits the proliferation of RA-resistant leukemic cell lines HL-60R and K562 but also induces apoptosis in these cells. Exposure of HL-60R to CD437 results in the rapid (within 30 minutes) increase of the cyclin-dependent kinase inhibitor p21(waf1/cip1) as well as GADD45 mRNA. Manifestations of CD437-mediated programmed cell death are noted within 2 hours, as indicated by both the cleavage and activation of the CPP32 protease and cleavage of poly (ADP-ribose) polymerase. This is followed by cleavage of bcl-2 and internucleosomal DNA degradation. HL-60R cells do not express the retinoid nuclear receptor RAR beta and RAR gamma and express a truncated RAR alpha. Thus, CD437 induction of p21(waf1/cip1) and GADD45 mRNAs and apoptosis occurs through a unique mechanism not involving the retinoid nuclear receptors. CD437 represents a unique retinoid with therapeutic potential in the treatment of myeloid leukemia.

Bcl-X(L) expression and its downregulation by a novel retinoid in breast carcinoma cells.

We have recently found a novel retinoid, 6-[3-(1-adamantyl)-4-hydroxphenyl]-2-naphthalene carboxylic acid (CD437), which induces G1 cell cycle arrest and apoptosis in human breast carcinoma (HBC) cells (Oncogene 11, 493-504, 1995). CD437 downregulates the expression of a number of proteins which antagonize apoptosis. bcl-X(L), a homologue of bcl-2, antagonizes apoptosis, while bcl-X(S) enhances apoptosis. We have found that estrogen receptor (ER)-negative HBCs express higher levels of bcl-X(L) and significantly lower levels of bcl-2 than their ER-positive counterparts. Neither cell type expresses bcl-X(S). The addition of CD437 (1 microM) results in a fourfold downregulation of bcl-X(L) mRNA and protein levels followed by apoptosis in MDA-MB-231 and MDA-MB-468 cells. CD437 concentrations as low as 10 nM cause a significant reduction in both bcl-X mRNA and bcl-X(L) protein expression. CD437-dependent downregulation of bcl-X mRNA and bcl-X(L) protein expression occurs within 24 h of CD437 addition to the cells. Retinoic acid does not effect bcl-X mRNA or bcl-X(L) protein expression. CD437 is a potent inducer of apoptosis in a number of breast carcinoma cells lines and downregulates the expression of a number of proteins which antagonize apoptosis.

Elevated expression of retinoic acid receptor-alpha (RAR alpha) in estrogen-receptor-positive breast carcinomas as detected by immunohistochemistry.

Retinoids modulate gene activity, cell growth and differentiation by binding to a series of nuclear receptors, i.e., retinoic acid receptors (RARs) or retinoid X receptors. Retinoic acid (RA) inhibition of estrogen receptor (ER)-positive breast carcinoma seems to be mediated through RAR alpha. Estrogens upregulate RAR alpha in ER-positive breast carcinoma cell lines. In this study we examined RAR alpha expression in the ER-positive MCF7 and ER-negative MDA-MB-231 human breast carcinoma cell lines as well as in 10 ER-negative and 9 ER-positive infiltrating ductal breast carcinoma specimens using immunohistochemistry and quantitation by image cytometry. MCF7 cells expressed twofold higher levels of RAR alpha protein than MDA-MB-231 cells. RAR alpha expression, as detected by immunostaining and quantitated by image cytometry, was upregulated in these cells by estradiol. ER-positive breast carcinoma specimens also exhibited approximately two-fold higher RAR alpha levels than their ER-negative counterparts. Thus, RAR alpha expression is significantly elevated in ER-positive breast tumors as assessed by detection and quantitation using immunohistochemical staining and image cytometry, respectively. Whether the decrease in RAR alpha protein levels and loss of RA-mediated growth inhibition in ER-negative tumor plays a role in the increased metastatic potential of ER-negative tumors remains to be determined.

Posttranscriptional regulation of p21WAF1/CIP1 expression in human breast carcinoma cells.

p21WAF1/CIP1 plays a major role in the induction of G1 arrest following DNA damage. Although p21WAF1/CIP1 expression is regulated by the tumor suppressor p53, induction of p21WAF1/CIP1 expression through p53-independent pathways has been described in numerous cell types. In this report, we describe the mechanism by which the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induces p21WAF1/CIP1 in breast carcinoma cells possessing either a wild-type (MCF-7 cells) or mutated (MDA-MB-468 cells) p53. Exposure of MDA-MB-468 cells to this retinoid results in an approximately 10-fold increase in p21WAF1/CIP1 mRNA levels, whereas less than a 2-fold increase in p21WAF1/CIP1 gene transcription was observed as indicated by transient transfection experiments utilizing a p21WAF1/CIP1 promoter firefly luciferase reporter gene construct and nuclear run-off studies. We found similar results in the MCF-7 cells (Z-M. Shao et al., Oncogene, 11: 493-504, 1995). We have now found that while enhancing p21WAF1/CIP1 gene transcription minimally, this retinoid increases p21WAF1/CIP1 mRNA stability by 3-fold in both cell types. We also demonstrate that approximately 1.5 kb of the 3' untranslated region causes enhanced instability of p21WAF1/CIP1 mRNA. The retinoid-dependent increase in p21WAF1/CIP1 mRNA stability is accompanied by an increase in p21WAF1/CIP1 protein expression, as indicated by Western blot experiments utilizing anti-p21WAF1/CIP1 monoclonal antibody. This increase in p21WAF1/CIP1 is subsequently followed by the onset of programmed cell death in both cell types. Thus, CD437 is a novel retinoid which enhances p21WAF1/CIP1 mRNA levels through stabilization of the message regardless of the p53 status of the cell.

Regulation of the human retinoic acid receptor alpha gene in the estrogen receptor negative human breast carcinoma cell lines SKBR-3 and MDA-MB-435.

Estradiol-mediated enhancement of retinoic acid receptor alpha (RARalpha) expression in the estrogen receptor (ER)-positive human breast carcinoma (HBC) cells results in their sensitivity to RA-mediated growth inhibition (A. K. Rishi et al., Cancer Res., 55: 4999-5006, 1995). Most ER-negative HBCs are known to express lower levels of RARalpha and are resistant to RA-mediated inhibition of growth. We show that ER-negative SKBR-3 and MDA-MB-435 HBCs express approximately 2-fold higher levels of RARalpha isoform 1 mRNA when compared to the ER-negative MDA-MB-231 and MDA-MB-468 HBCs. SKBR-3 cells are sensitive to growth inhibition by RA, and by using RARalpha-selective synthetic retinoids, we demonstrate that the antiproliferative effects of RA in the SKBR-3 cell line are accomplished, in part, via activation of RARalpha. Both MDA-MB-231 and MDA-MB-468 HBCs are not growth inhibited by RA or any of the retinoids tested. Transient transfection experiments using a 5.0-kb RARalpha promoter fragment fused to the luciferase reporter gene showed 2-3-fold higher transcriptional activation in SKBR-3 cells when compared to MDA-MB-468 cells. We report identification of a 72-bp fragment of RARalpha promoter that contains unique cis elements responsible for mediating an estradiol-independent 2.5-fold enhancement of RARalpha gene expression in SKBR-3 and MDA-MB-435 cells.

H19 gene overexpression in atypical multidrug-resistant cells associated with expression of a 95-kilodalton membrane glycoprotein.

The multidrug resistance phenotype of human breast carcinoma MCF-7/AdrVp cells is characterized by overexpression of a 95-kilodalton membrane glycoprotein (p95), accompanied by a marked reduction in intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein. We discovered that the mRNA of the H19 gene is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells or drug-sensitive MCF-7/AdrVp revertant cells. H19 is an imprinted gene with an important role in fetal differentiation, as well as a postulated function as a tumor suppressor gene. Another p95-overexpressing multidrug-resistant cell line, human lung carcinoma NCI-H1688, also displays high levels of H19 mRNA. In contrast, several multidrug-resistant cell lines that overexpress P-glycoprotein or the multidrug resistance protein do not have higher levels of H19 mRNA than their drug-sensitive counterparts. This is the first report of H19 gene overexpression accompanying any form of drug resistance. The association of H19 and p95 gene expression in drug resistance warrants further study.