Synergistic antiproliferative effect of human recombinant interferons and retinoic acid in cultured breast cancer cells.

A combination of retinoic acid (RA) and human recombinant DNA-derived interferon-gamma (Hu-IFN-gamma) was tested with respect to the growth inhibitory action on several human mammary carcinoma cell lines (ZR-75.1, 734-B, MCF-7, and BT-20), a human lung carcinoma cell line (CCL-185), and a human laryngeal carcinoma cell line (HEP-2). The mammary carcinoma cell lines were all sensitive to Hu-IFN-gamma, and 2 of them (ZR-75.1 and 734-B) were also affected by RA. The combination of both substances led to a pronounced synergistic amplification of growth inhibition in ZR-75.1 and 734-B cells. RA also increased the antiproliferative activity of Hu-IFN-gamma in the RA-resistant BT-20 cells and to a less pronounced degree in MCF-7 cells. In contrast to these findings, no synergistic effects were observed between Hu-IFN-gamma and RA in CCL-185 and HEP-2 cells. Human recombinant DNA-derived interferon-alpha 2 amplified the action of RA only in BT-20 cells, but it did not act synergistically with RA in the other cell lines tested.

Effect of 4-hydroxyphenylretinamide and retinoic acid on proliferation and cell cycle of cultured human breast cancer cells.

The synthetic retinoid 4-hydroxyphenylretinamide (HPR) showed antiproliferative effect on cultured human breast cancer cells, which were sensitive to retinoic acid (RA) too. Investigation of the cell cycle by flow cytophotometry showed a significant increase of cells in the S-phase of the cell cycle after 24 hours of RA treatment, whereas HPR did not show this effect. After 4-7 days of retinoid treatment, the percentage of resting cells increased. The influence on the cell cycle and the antiproliferative effect were more pronounced for RA than for HPR treatment in both cell lines investigated (ZR-75.1 and 734 B).

Methylation and silencing of the retinoic acid receptor-beta2 gene in breast cancer.

BACKGROUND: A growing body of evidence supports the hypotheses that the retinoic acid receptor beta2 (RAR-beta2) gene is a tumor suppressor gene and that the chemopreventive effects of retinoids are due to induction of RAR-beta2. RAR-beta2 expression is reduced in many malignant tumors, and we examined whether methylation of RAR-beta2 could be responsible for this silencing. METHODS: RAR-beta2 expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in eight breast cancer cell lines that were either treated with the demethylating agent 5-aza-2'-deoxycytidine and subsequently with all-trans-retinoic acid (ATRA) or left untreated. Sodium bisulfite genomic sequencing was used to determine the locations of 5-methylcytosines in the RAR-beta2 genes of three of these cell lines. In 16 breast cancer biopsy specimens and non-neoplastic breast tissue, methylation-specific PCR was used to determine the methylation status of RAR-beta2, and, in 13 of the specimens, RT-PCR analysis was used to detect RAR-beta2 expression. RESULTS: Cell lines SK-BR-3, T-47D, ZR-75-1, and MCF7 exhibited expression of RAR-beta2 only after demethylation and treatment with ATRA. The first exon expressed in the RAR-beta2 transcript was methylated in cell lines ZR-75-1 and SK-BR-3. Six breast cancer specimens showed methylation in the same region of the gene. No expression of RAR-beta2 was found in any grade III lesion. An inverse association between methylation and gene expression was found in all grade II lesions. The RAR-beta2 gene from non-neoplastic breast tissue was unmethylated and expressed. CONCLUSIONS: Methylation of the RAR-beta2 gene may be an initial step in breast carcinogenesis; treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality.

Effects of retinoic acid and gamma-interferon on expression of retinoic acid receptor and cellular retinoic acid-binding protein in breast cancer cells.

Retinoids and gamma-interferon (IFN-gamma) have been demonstrated to synergistically amplify growth inhibition in cultured breast cancer cells. Since IFN-gamma enhances effects mediated by retinoids more than vice versa, we focused our investigations on the mRNA expression of cellular retinoic acid-binding proteins (CRABPs) and retinoic acid receptors (RARs), since these are the key molecules that mediate retinoid action. Synergistic inhibition of cell proliferation under treatment with 9-cis-retinoic acid and IFN-gamma was detected in the three breast cancer cell lines MCF-7, SKBR-3, and even in the RA-resistant BT-20 cells. RAR-alpha and RAR-gamma mRNA were observed in all cell lines, whereas RAR-beta was not detectable. CRABP I message was expressed in MCF-7 cells only, but CRABP II was found in all three breast cancer cell lines. IFN-gamma (10 ng/ml) increased RAR-gamma expression but had no influence on RAR-alpha, whereas RAR-beta was not detectable in any of the cell lines. RA (1 microM)-mediated CRABP II increase was suppressed by IFN-gamma (10 ng/ml). These observations indicate that IFN-gamma-mediated increase in RAR-gamma and suppression of RA-mediated CRABP II activation may play a role in synergistic inhibition of proliferation in breast cancer cell lines.

Modulation of ovarian carcinoma tumor marker CA-125 by gamma-interferon.

Interferons are known to modulate several cellular functions by the induction of different proteins. In our study a gamma-interferon-mediated presentation of one of the most important ovarian tumor markers (CA-125) on the cell surface was demonstrated using two ovarian carcinoma cell lines in vitro (HTB-77 and SKOV-3). This induction was found to be dependent on an intact protein biosynthesis. The gamma-interferon effect reached a maximum on the third day of treatment. Under such conditions the CA-125 concentration was increased intracellularly, on the cell surface, and in the supernatant culture medium. The surface antigen was shed rapidly with a half-life of 1 day. The addition of dexamethasone to gamma-interferon treated HTB-77 cells improved CA-125 expression synergistically. HLA-DR and CA-125 expression was found to be regulated by interferons in different ways. The demonstration of CA-125 expression provides an important insight into the potential regulatory mechanism governing this tumor marker. Since interferons are naturally occurring substances as well as therapeutically administered agents, it seems necessary to pay attention to possible tumor marker modulation.

Effects of human recombinant alpha 2 arg-interferon and gamma-interferon on human breast cancer cell lines: dissociation of antiproliferative activity and induction of HLA-DR antigen expression.

Human recombinant gamma-interferon (rhu-IFN-gamma) and human recombinant alpha-interferon (rhu-IFN-alpha 2 arg) with a chemical purity of over 95% were compared for their antiproliferative and HLA-DR-inducing activity in five human breast cancer cell lines (BT 20, ZR 75.1, MCF 7, 734B, Hs578T). Cytostatic effects on tumor cells were evaluated in monolayer cultures. HLA-DR antigen expression was examined by an indirect immunofluorescence technique using two different anti-HLA-DR monoclonal antibodies (anti-HLA-DR, VID-1) against framework determinants. rhu-IFN-gamma and rhu-IFN-alpha 2 arg differed in their antiproliferative efficiency in terms of both dose dependency and the spectrum of sensitive target cells. Combinations of rhu-IFN-gamma and rhu-IFN-alpha 2 always resulted in higher cytostatic effects. HLA-DR expression was exclusively inducible by rhu-IFN-gamma and did not correspond to its antiproliferative activity. Furthermore, HLA-DR expression did not depend on proliferation but did require intact RNA and protein syntheses as shown by inhibition with cycloheximide and actinomycin D. HLA-DR antigen expression in mammary cancer lines was dependent on time, dose, and the continued presence of rhu-IFN-gamma. Thus, our data suggest that in particular combinations type I and type II interferons might be useful in the treatment of breast cancer because they provide effective cytostatic and cell membrane-modulating properties.

Paclitaxel- and docetaxel-dependent activation of CA-125 expression in human ovarian carcinoma cells.

Taxanes represent a new class of antineoplastic agents that are being evaluated in several malignant tumors; they have been shown to induce a high remission rate and to prolong survival in ovarian cancer patients. However, CA-125 has been suggested to be an unreliable marker for monitoring response to paclitaxel therapy. Therefore, we were interested in whether taxanes may directly modulate CA-125 expression. Human ovarian carcinoma cell lines OVCAR-3, HOC-7, SKOV-6, 2780, 2774, and HTB-77 were treated with paclitaxel or docetaxel. Secreted, surface-associated, and cytosolic CA-125 were estimated by means of a sandwich solid-phase RIA or by immuno-flow cytometry. In addition to in vitro antiproliferative activity, paclitaxel and docetaxel augmented the expression of the tumor marker CA-125 in the three ovarian carcinoma cell lines, OVCAR-3, HOC-7, and SKOV-6, constitutively expressing this tumor marker. The three CA-125-negative cell lines, 2780, 2774, and HTB-77, did not respond to taxane treatment by expressing this tumor marker, although their proliferation was markedly inhibited. The taxane-mediated induction of CA-125 was found to be dependent on intact protein and RNA biosynthesis. However, CA-125 concentration was increased in the supernatant medium only and not on cell surface or cytosol. Our results demonstrate an in vitro activation of ovarian carcinoma cells in terms of CA-125 secretion by taxanes. This may explain the CA-125 fluctuations observed in vivo under paclitaxel treatment and may indicate that CA-125 is not a reliable tumor marker during taxane chemotherapy.

Loss of retinoic acid receptor beta expression in breast cancer and morphologically normal adjacent tissue but not in the normal breast tissue distant from the cancer.

Retinoids and their receptors [retinoic acid receptors (RARs) and retinoid X receptors] play an important role in maintaining the balance between proliferation and apoptosis. Recently, Deng et al. [Science (Washington DC), 274: 2057-2059, 1996] reported a loss of heterozygosity on chromosome 3p24 in breast cancer specimens and the morphologically normal appearing adjacent tissue. The 3p24 locus includes, among other genes, the region coding for RAR-beta. This study was designed to determine whether there are abnormalities in the expression of retinoid receptors in surgical specimens of patients with breast cancer. In 14 patients, transcripts of nuclear retinoid receptors were detected by in situ hybridization in formalin-fixed, paraffin-embedded specimens by means of digoxigenin-labeled riboprobes specific for RAR-alpha, -beta and -gamma. We found RAR-alpha expressed in all specimens, whereas RAR-gamma was expressed in 100% of normal breast tissue but in only 11 of 14 tumorous lesions. RAR-beta was found in all cases of normal breast tissue localized distant from the tumor, but in 13 of 14 cases it was completely absent in the tumor and the morphologically normal appearing tissue adjacent to the tumor. One possibility to explain the suppression of RAR-beta is a mutation in the promoter region. Sequencing the DNA extracted from paraffin-embedded tumor tissue of the corresponding breast cancer specimens, we were not able to detect any mutation in the retinoic acid-responsive element. Our results clearly indicate a crucial role of RAR-beta in the carcinogenesis of breast cancer.

Gamma-interferon reduces expression of the protooncogene c-erbB-2 in human ovarian carcinoma cells.

The overexpression of the protooncogene c-erbB-2 (HER-2/neu) in ovarian and mammary carcinoma is an important indicator for a bad prognosis. In this study we demonstrate that, in three of four ovarian carcinoma cell lines, there is a gamma-interferon-mediated reduction in c-erbB-2 specific protein, and this effect was found to correlate with the antiproliferative action. It is interesting to note that there is no relation between the absolute amount of c-erbB-2 protein expressed and the sensitivity of the ovarian carcinoma cells for an antiproliferative activity of gamma-interferon. Other chemotherapeutic agents did not affect c-erbB-2 expression, although they inhibited the proliferation. The oncogene expression was lowered only in the ovarian carcinoma cell lines and not in three gamma-interferon-sensitive human breast cancer cell lines. Expression of the oncogene c-erbB-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which gamma-interferon inhibits cell proliferation.

Ascitic interleukin-12 is an independent prognostic factor in ovarian cancer.

PURPOSE: The clinical impact of endogenous cytokines supplied with deterministic properties in the generation of either T helper (Th)1 -type or Th2-type immune response was investigated in patients with ovarian cancer. Whereas interleukin (IL)- 12 initiates the differentiation of naive Th0 cells toward Th1 phenotype, IL-4 and IL-10 mediate the development of Th2-type immunity. PATIENTS AND METHODS: Cytokines were determined before treatment by means of enzyme-linked immunosorbent assay (ELISA) in ascites fluid and serum of 76 patients with ovarian cancer. Cytokine levels were compared with each other and with standard clinicopathologic parameters. A stepwise logistic regression was calculated to rule out interdependence in the associations of the various variables. Survival analyses were performed with the Kaplan-Meier method and differences in survival were examined according to Mantel and Breslow. Cox proportional hazards analysis was used to identify independent prognostic factors. RESULTS: Whereas IL-10 and IL-12 were detectable in all ascites-fluid samples, IL-4 was measurable in only 43% of the specimens. With the exception of neopterin, macrophage colony-stimulating factor (M-CSF), and IL-4, determined cytokine levels were significantly elevated in ascites fluid compared with serum (P < .01). In univariate analyses, high ascitic-fluid concentrations of either neopterin, tumor necrosis factor-alpha (TNF-alpha), or IL-12 were associated with poor disease-free (P < .005) and overall (P < .01) survival. Multivariate Cox regression analysis showed ascitic-fluid IL-12 levels to be the only immunologic variable that retained independent prognostic significance (P < .03 for disease-free and P < .01 for overall survival), together with residual disease, Fédération Internationale de Gynécologie et d'Obstétrique (FIGO)-stage, and patient age. CONCLUSION: In ovarian cancer, high ascitic-fluid IL-12 levels, which may indicate a local Th1-generated immune response, are associated with disease progression.

Detection of a heat- and acid-stable 'progesterone'-binding protein in the rat lung.

Using a modified charcoal method, we could detect a steroid-binding component in rat lung cytosol which specifically binds R5020, progesterone, and some of its natural derivatives. The concentration of binding sites is high (30-40 pmol/mg protein), the affinity is moderate, the Kd of the R5020 complex being approximately 10(-7) M. Proteolytic enzymes and sulfhydryl reagents destroyed the binding sites indicating the protein nature and the requirement for disulfide bonds. The protein sedimented in the 2 S range thus had an Mr of 10,000-15,000. Further characteristics are the extreme heat (30 min at 100 degrees C) and acid (pH 1) stability. These properties and the fact that it was not detected in serum, distinguish this binding protein from receptors and specific serum steroid binders.

Modulation of tumour marker CA-125 expression in cultured ovarian carcinoma cells.

The aim of this study was to elucidate whether proliferation of ovarian carcinoma cells may affect the biosynthesis and release of CA-125. In a cell culture model the tumour marker CA-125 expression in cytosol, surface membrane, and release into culture medium was studied in six human carcinoma cell lines. Cell cycle analysis of propidium iodide stained nuclei was performed using a fluorescent activated cell sorter. The turnover of CA-125 is very rapid, within 24 h the equivalent amount found in each cell was also released in the supernatant culture medium. A good relation between cytosolic, membrane, and released CA-125 was observed. CA-125 expression was associated predominantly with the G0/G1 phase of the cell cycle and was dependent on cell density. The results presented here demonstrate that factors associated with tumour cell proliferation could influence the CA-125 serum level in ovarian cancer patients.

The prognostic value of nuclear roundness and neopterin in ovarian cancer.

The prognostic value of clinical factors, morphometric features and neopterin, a marker for macrophage activation, was investigated retrospectively in 68 ovarian carcinoma patients. Nuclear roundness was a good predictor of patient survival. About 50% of our patients showed neopterin concentrations above the cut-off level of 275 mumol/mol creatinine. Interestingly, those patients with elevated urinary neopterin concentration, and thus displaying a sign of activation of cell-mediated immunity, had a shorter survival than those with normal concentration. Applying a multivariate Cox regression analysis, the only independent parameters predicting patient survival were FIGO stage, residual disease, nuclear roundness and neopterin.

Retinoic binding in melanomas of the eye and in normal choroid.

Binding proteins for retinoic acid (cellular retinoid acid binding protein, CRABP), and for vitamin A (cellular retinol binding protein, CRBP) have been demonstrated in various cell types; these binding proteins display the characteristics of receptors. In the present study CRABP and CRBP levels were measured in 9 melanomas of the choroid. CRABP was detected in 2 of the melanomas, whereas CRBP was measurable in 1 melanoma. In comparison samples of normal choroid contained CRABP and CRBP in all cases investigated.

The role of polyamines in interferon and retinoic acid mediated synergistic antiproliferative action.

Retinoic acid alone has no effect on the human breast cancer cell line BT-20 but can amplify the antiproliferative action of interferon-gamma (IFN-gamma). In our system ornithine decarboxylase (ODC) activity correlates well with growth rate; it was investigated whether the antiproliferative effects of IFN-gamma and IFN-gamma plus retinoic acid could be attributed to suppression of ODC activity. The ODC inhibitor difluoromethylornithine (DFMO), which is active as a single agent did not enhance growth inhibition induced by the biological response modifiers. The substitution of the BT-20 cells with putrescine, the product of the enzymatic reaction mediated by ODC, reversed DFMO induced antiproliferative action. On the other hand putrescine did not affect the proliferation of BT-20 cells treated with interferon alone or in combination with retinoic acid.

Transforming growth factor-beta and ovarian carcinoma cells: regulation of proliferation and surface antigen expression.

Transforming growth factor-beta (TGF-beta) is a multifunctional peptide regulating several processes in ovarian cells. The growth of ovarian carcinoma cell lines (OVCAR-3, HTB-77, 2780 and CRL-1572) was reduced by TGF-beta in a dose related manner. The antiproliferative activity was not improved by combination with other biological response modifiers. Treatment with TGF-beta augmented the expression of interferon-gamma induced class I and II antigens of the major histocompatibility complex. The presentation of another antigen namely the tumor marker CA-125 on the cell surface was markedly reduced by TGF-beta.

Human interferon-gamma increases adhesion of cultured carcinoma cells to the substratum.

Effects of human recombinant-DNA derived interferon-gamma and -alpha 2 on the adhesion of cultured breast cancer cells (BT-20, ZR-75.1, MCF-7, 734-B and Hs-578-T), larynx carcinoma cells (HEP-2), epidermoid carcinoma cells (KB), lung carcinoma cells (CCL 185), and ovarian carcinoma cells (1847) to the surface of cell culture plastic dishes were studied. Layered cells were detached after a 3-day treatment with interferon either by trypsin-EDTA, trypsin, protease or cooling to 4 degrees C. Treatment with interferon-gamma (500 unit/ml) significantly increased the incubation time for trypsin-EDTA, EDTA and at 4 degrees C necessary to bring cells into suspension for the 4 cell lines BT-20, ZR-75.1, MCF-7 and HEP-2. Interferon-alpha 2 was not able to induce a similar effect. Reattachment of interferon-gamma treated ZR-75.1 cells was not increased after harvesting by trypsinization or EDTA action. Decreased adhesion of cultured cells is associated with transformation and the effects of interferon-gamma may be explained by reinforced normal phenotype. Interferon-gamma induced adhesion was not associated with other interferon effects especially the anti-proliferative activity or modulation of surface antigens.

Effect of retinoic acid and 4-hydroxytamoxifen on human breast cancer cell lines.

Using established breast cancer cell lines in a cell culture model we studied the growth effect of retinoic acid (RA) alone or in combination with the antiestrogen 4-hydroxytamoxifen (OHT). Cytoplasmic 3HRA binding sites were determined by sucrose density gradient centrifugation analysis. Of the three cell lines Hs578T, BT 20, and 734 B only the last showed a significant amount of specific RA binding (10(5) sites/cell). This cell line showed a dose dependent decrease in proliferation after a long-term incubation with RA whereas the 3H-thymidine uptake was highly significantly increased after incubation with 10(-6)M of RA for 20 hr. Growth inhibition was not further increased by the addition of OHT (10(-6) M), but the increase in thymidine incorporation due to RA was neutralized by OHT. Hs578T and BT 20 cells were not affected by any of the treatments. The different action of RA on proliferation and thymidine incorporation suggests a cell cycle specific mechanism.

Inhibition of the estradiol-induced growth of cultured human breast cancer cells by the anti-estrogens tamoxifen, desmethyl-tamoxifen, 4-hydroxy-tamoxifen and enclomiphene.

The growth effects of tamoxifen (T), desmethyl-tamoxifen (dMeT), 4-hydroxy-tamoxifen (OHT) and enclomiphene (Clo) on cultured human breast cancer cell lines have been related to published binding affinities for the estrogen receptor. Only in cells which were stimulated by estrogens did these anti-estrogens markedly inhibit growth. In both estrogen sensitive cell lines tested, 734 B and ZR 75.1, the anti-estrogen activity showed the identical rank: OHT much greater than Clo approximately equal to T = dMeT; this anti-proliferative potency agrees with reported affinities of these compounds for the estrogen receptor. In culture media containing defined amounts of estradiol we observed that a 10,000-fold molar excess of OHT was required to inhibit the estradiol-induced growth, but the estradiol-independent proliferation was not affected.

New insights into p53 regulation and gene therapy for cancer.

Due to its critical involvement in cell cycle control and apoptotic signaling, the transcription factor p53 has become the most important tumor suppressor currently under investigation. TP53 is the most frequently mutated gene in human cancers and is thought to play a crucial role in malignant transformation. Therefore, p53 appears to be an appealing target for gene therapy. Adenoviral-based p53 gene transfection is now being introduced in large clinical trials. Viral cell entry was found to be the rate-limiting step of gene delivery and thus of therapeutic efficiency. Attachment of adenoviruses to the target cell surface is mediated through the coxsackie-adenovirus receptor, and internalization is achieved via interactions with integrins of the alpha v beta(3) and alpha v beta(5) class. The assumption that the restitution of the p53-dependent apoptotic pathway results in a higher responsiveness of solid tumors to cytostatic agents remains a major matter of debate. Combinations of p53-based gene therapy with other components involved in apoptosis, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/APO2L, or agents neutralizing tumor-promoting antiapoptotic signals, such as humanized anti-growth factor antibodies, should further improve the effectiveness of cancer treatment in the future.