Inhibition of growth-factor-activated proliferation by anti-estrogens and effects on early gene expression of mcf-7 cells

Recently, it was reported that the anti-estrogen tamoxifen not only inhibits estradiol-stimulated growth of MCF-7 cells but also significantly reduces the proliferation rate of cells stimulated by growth factors. We have confirmed this finding and also shown that the new anti-estrogen droloxifene inhibits the proliferation of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I)-stimulated MCF-7 cells. The growth-factor-induced proliferation was inhibited in a dose-dependent manner by the anti-estrogens in the complete absence of estrogen and FCS. Of the anti-estrogens, droloxifene was considerably more potent than tamoxifen. Because the exprersion of the proto-oncogenes c-fos and c-myc has been considered a key event in development of the mitogenic response, we examined the effects of anti-estrogens on c-myc and c-fos gene expression. We included in these investigations the steroidal anti-estrogen ICI 164,384 because this compound has no or very little estrogenic activity. The studies revealed that all 3 antiestrogens transiently induced c-myc mRNA expression. However, the anti-estrogens inhibited estradiol-induced c-myc mRNA expression, although with different potencies. Pre-incubation of MCF-7 cells with droloxifene and tamoxifen resulted in elevated levels of growth-factor-induced c-myc mRNA expression. In contrast, the anti-estrogens did not induce c-fos mRNA or affect the expression of c-fos mRNA induced by growth factors. In conclusion, non-steroidal anti-estrogens inhibit growth-factor-stimulated proliferation of MCF-7 cells without inhibitinggrowth-factor-induced c-myc or c-fos mRNA expression.     

Effects of an inducible anti-sense c-myc gene transfer in a drug-resistant human small-cell-lung-carcinoma cell line

Small-cell-lung-cancer (SCLC) is characterized by rapid development of resistance to cytotoxic agents, such as cisplatin (cDDP) and anthracyclines. c-myc over-expression is one of the reported genetic alterations in this tumor. Amplification of the c-myc gene in this and other cancers is often correlated with poor prognosis. We studied the existence of a correlation between resistance and activation of the c-myc oncogene in a cDDP-resistant SCLC sub-line, GLC₄cDDP, containing a c-mycgene amplification. This cell line was stably transfected with either an anti-sense c-myc (AS) or control (C) expression vector resulting in the sub-clones GLC₄cDDP/AS and GLC₄cDDP/C respectively. PCR and RT-PCR analysis illustrated the integration and activation of the dexamethasone(dex)-inducible MMTV-LTR promoter linked to the complete AS-c-myc fragment in GLC₄cDDP/AS cells, but not in GLC₄cDDP/C cells. Dex-induced AS-c-myc RNA resulted in 50% growth inhibition during the first 48 hr, which declined to 25% at 72 hr. In addition, AS-c-myc RNA expression reduced the cloning efficacy by 36% and induced 2-fold more apoptosis within 24 hr in GLC₄cDDP/AS cells. Dex treatment did not affect the proliferation, clonogenicity and induction of apoptosis in the control cell lines. Furthermore, AS-c-myc RNA expression caused a 1.4-fold increased cDDP sensitivity but no change in doxorubicin or vincristine sensitivity in GLC₄cDDP/AS cells. Our results indicate that AS-c-myc RNA expression causes inhibition of cell proliferation, induces apoptosis, reduces clonogenicity and interferes with cDDP sensitivity but not with doxorubicin or vincristine sensitivity. Int. J. Cancer73:544–550, 1997. © 1997 Wiley-Liss, Inc.     

1,25-dihydroxyvitamin D3 and retonic acid analogues induce differentiation in breast cancer cells with function- and cell-specific additive effects

Vitamin D₃ derivatives and retinoids can induce cell cycle arrest, differentiation and cell death in many cell lines. These compounds can act cooperatively in some of their functions and may be of potential use either individually or in combination in the treatment of breast cancer. The effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), all-trans retinoic acid (ATRA) and several analogues were evaluated on malignant phenotypic traits of breast cancer cell lines MCF-7, T-47D and MDA-MB-231. Both 1,25(OH)₂D₃ and ATRA caused a decrease in anchorage independent colony formation in MCF-7 and T-47D cells in a dose-dependent manner. The effects of 1,25(OH)₂D₃ 10^–10 and 10^–9M were synergistic with ATRA 10^–8M in T-47D cells but were antagonistic in both MCF-7 and in T-47D cells at most concentrations. Both 1,25(OH)₂D₃ and ATRA individually induced an accumulation of MCF-7 cells in the G₁ phase of the cell cycle and an associated increase in p21^WAF1/Cip1, p27^Kipl and a dephosphorylation of Rb but the effects were not additive. Both compounds inhibited the invasive capacity of MDA-MB-231 cells. 1,25(OH)₂D₃ but not ATRA caused an increase in E-cadherin levels in MDA-MB-231 cells. These two functions were not additive. The compounds 1,25(OH)₂D₃, a noncalcemic analogue 1,25(OH)₂-16-ene-23-yne-D₃, ATRA, AGN195183, an RAR-specific agonist, and AGN190168 (tazarotene), an RAR-selective agonist, induced differentiation as determined by measurements of lipid droplet formation. The individual effects of 1,25(OH)₂-16-ene-23-yne-D₃ combined with ATRA or with tazarotene at 10^–9M each were additive in MCF-7 and MDA-MB-231 cells on lipid formation. The data demonstrate that both 1,25(OH)₂D₃, ATRA, and selected analogues induce a more differentiated phenotype in breast cancer cells with additive effects that are function- and cell-specific.     

Lymphoblastoid cells transfected with c-myc: Downregulation of EBV-lytic antigens and impaired response of autologousCD4+ T cells in vitro

Normal EBV-positive lymphoblastoid B-cell lines (LCL) were transfected with vectors containing the c-myc oncogene (pHEBO-Eμ- myc) or control vectors (pHEBO-Eμ) and analyzed for the expression of EBV-lytic and latent antigens. While EBV-latent antigens were normal in the c-myc transfectants there was an almost complete downregulation of EBV-lytic antigens, including BZLFI, EA(D), gp340 and VCA. These observations were consistently repeated on 6 different LCLs transfected with c-myc. Unlike control LCLs, the c-myc transfectants did not release infectious EBV. PCR analysis demonstrated that BZLFI mRNA was virtually absent in c-myc transfectants, possibly suggesting that the deregulated c-myc imposed a block in the EBV-lytic cycle at this particular level. c-myc transfectants failed to sustain the proliferative response of autologous CD4⁺ T-cell clones with specificity for EBV-lytic antigens. However, they regained this capacity after incubation with ultraviolet-inactivated EBV or gp340 antigen in vitro, also indicating that their antigen-presenting capacities were not impaired. c-myc transfectants failed to elicit a secondary proliferative response by autologous CD4⁺ T cells purified from the peripheral blood of EBV-seropositive donors. Exposure of c-myc transfectants to UV-inactivated EBV again resulted in a proliferative CD4⁺ T-cell response comparable to that elicited by the control LCLs. Collectively, our data provide evidence for the remarkable ability of an oncogene to influence the life cycle of a virus and to modify the antigenicity of the infected cells. © 1996 Wiley-Liss, Inc.     

Investigation of the cooperative effects of transforming growth factor α and c-myc overexpression in rat liver epithelial cells

Overexpression of both transforming growth factor (TGF)-α and c-myc is consistently reported in hepatic tumors. We transfected rat liver epithelial cells (RLECs) with expression vectors for TGF-α, c-myc, or both and analyzed the morphology, biological properties, and tumorigenicity of clones that overexpressed these genes. The transfectants were morphologically indistinguishable from the parental RLECs, but the overexpression of TGF-α resulted in changes in growth properties and an enhanced response to the mitogenic effects of hepatocyte growth factor. The concomitant overexpression of c-myc decreased growth factor requirements of the TGF-α/c-myc clones compared with RLEC and TGF-α clones. The TGF-α and TGF-α/c-myc clones were tumorigenic in nude mice at frequencies of 27% and 53%, respectively, indicating that the genes cooperate in malignant transformation. However, the untransformed nature and low tumorigenicity of the transfectants suggest that transformation depends on other cellular events in addition to the overexpression of TGF-α or c-myc. Characterization of tumor cell lines showed that in contrast to the transfectants, the tumor clones were morphologically transformed, capable of autonomous growth and anchorage-independent growth, and aggressively tumorigenic with a frequency of 100%. Clearly, the tumor cells differed from the transfectants and had undergone biological or genetic alterations (or both) as a consequence of the overexpression of TGF-α or c-myc. Our data suggest that the overexpression of TGF-α leads to enhanced responsiveness to hepatocyte growth factor, whereas the concomitant overexpression of c-myc confers growth-factor independence, providing a potential explanation of the mechanisms by which the overexpression of these genes results in transformation. © 1995 Wiley-Liss, Inc.     

c-myc amplification in pre-malignant and malignant lesions induced in rat liver by the resistant hepatocyte model

We have investigated by restriction fragment analysis genomic abnormalities involving the c-myc gene in DNA isolated from adenomas and hepatocellular carcinomas (HCCs). Adenomas and HCCs were induced by the “resistant hepatocyte” protocol in diethylnitrosamine-initiated male F344 rats. Southern-blot analysis of EcoRI-restricted DNA from normal liver, early and late adenomas, 12 weeks (EAs) and 30 weeks (LAs) after initiation, and HCCs, showed 2 bands of 18 and 3.2 kb hybridizing with c-myc, in all tissues. c-myc amplification occurred in almost all HCCs, and in the majority of EAs and LAs. These results were confirmed by dilution analysis. c-myc amplification was also seen in adenomas and HCCs by Southern analysis with HindIII-restricted DNA, and in HCCs by differential PCR. c-myc mRNA increase occurred in all adenomas and HCCs, but it was higher in the lesions showing gene amplification. Moreover, a 13-kb DNA extraband, hybridizing with c-myc, was found in the HindIII-restricted DNA from HCCs, but not in normal liver and adenomas, and a 7.1-kb extra band was present in EcoRI-digested DNA from one LA. EcoRI-restricted DNA from some adenomas exhibited a decrease in intensity of the 18-kb fragment, and an increase in intensity of the 3.2-kb fragment. No alteration in banding pattern occurred in the β-actin gene in adenomas. These results provide evidence of amplification and some other rearrangements involving the c-myc gene, in pre-malignant and malignant liver lesions, induced by the RH protocol, and suggest a role of c-myc rearrangement in the progression of adenomas to malignancy. © 1996 Wiley-Liss, Inc.     

Tumorigenic activity of a rearranged c-myc gene from a human T-cell leukemia line

The T-lymphoma cell line Hut78 contains a rearranged c-myc oncogenederived from a translocation between the long arms of chromosomes8 and 2; the event deletes the 3' end of the gene, causing theloss of the transcribed AT-rich sequence. It has recently beenshown that the mutant c-myc mRNA is several-fold more stablethan normal c-myc mRNA. We have assessed the tumorigenicityof the mutant c-myc allele by transfecting this gene and itsnormal counterpart into NIH3T3 cells, together with a neomycinresistance gene. Following selection for G-418 resistance, thecells were injected into nude mice. Tumors containing integratedc-myc arose in animals injected with cells transfected by themutated, but not by the normal, allele. The results suggestthat this rearranged c-myc bears a tumorigenic activity notobserved in other naturally occurring mutated c-myc allelesand may have directly contributed to the twuorigenic event inthe Hut78 cell line.     

C15-functionalized 16-ene-1α,25-dihydroxyvitamin D3 is a new vitamin D analog with unique biological properties

The Δ(16) structure as a vitamin D analog enhanced vitamin D receptor (VDR) binding affinity and induced significant cell differentiation, whereas its relative calcemic activity was reduced compared to 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)). Methodologies available to introduce a double bond at C16-C17 of the D-ring on the seco-steroidal skeleton were limited; therefore, a new synthetic strategy was developed to obtain not only the Δ(16) structure, but also a new C15-functional group. Since C15-functionalization was unprecedented in vitamin D analog studies, the hybrid structure of Δ(16) and the C15-OH group at the D-ring may provide important information on the structure-activity relationship with vitamin D analogs. The synthesized 16-ene-2α-methyl-1α,15α,25-trihydroxyvitamin D(3) showed almost 3-times higher VDR binding affinity and an equipotent level of osteocalcin promoter transactivation activity in human osteosarcoma cells as compared to 1α,25(OH)(2)D(3).     

Upregulation of endogenous p53 and induction of in vivo apoptosis in B-lineage lymphomas of Eμ-myc transgenic mice by deregulated c-myc transgene

Eμ-myc transgenic mice carry a constitutively overexpressed c-myc oncogene and develop B-lineage lymphomas. Previous studies have shown that c-myc overexpression can lead to in vitro apoptosis. Here, we investigated the in vivo effects of altered c-myc expression on cell proliferation versus death in spontaneously arising Eμ-myc tumors. Eμ-myc tumors display extensive in vivo apoptosis confined to small clusters of cells with greatly increased expression of both the c-myc transgene and the endogenous p53 gene as compared with that in normal, pretumor, or surrounding tumor tissue. This restricted overexpression of both the c-myc transgene and the endogenous p53 gene in small clusters of apoptotic tumor cells indicates that overexpression of these genes and apoptosis are not obligatory or uniform during tumor development and suggests that further somatic mutations or microenvironmental influences may be responsible for these properties. Nevertheless, the clear ability of tumor cells to undergo apoptosis in vivo may be exploitable for therapeutic purposes. Mol. Carcinog. 18:66–77, 1997. © 1977 Wiley-Liss, Inc.     

The preliminary application of in situ hybridization in detecting proto-oncogenes expression in human leukemic cells

An in situ hybridization technique with³⁵S labelled proto-oncogene probes (c-myc & c-fes) was used to detect their expression in bone marrow cells of 22 cases of leukemia of various types and immature granulocytes and erythroblasts of 16 nomal myelograms as controls. Both c-myc and c-fes were detectable in leukemic cells as well as in immature granulocytes and erythroblasts of normal bone marrow, but the expression extent varied in different cases. The levels of c-myc expression in leukemic cells were higher than those in controls (P<0.001). There was no difference of c-fes expression in two groups of bone marrow cells (P>0.05). This technique provides us a new method in studying variations of proto-oncogene expression in leukemic cells.     

1α,25-Dihydroxyvitamin D3 Modulates CYP2R1 Gene Expression in Human Oral Squamous Cell Carcinoma Tumor Cells

Oral squamous cell carcinomas (OSCC) are the most common malignant neoplasms associated with mucosal surfaces of the oral cavity and oropharynx. 1α,25-Dihydroxyvitamin D3 (1,25(OH)₂D3) is implicated as an anticancer agent. Cytochrome P450 2R1 (CYP2R1) is a microsomal vitamin D 25-hydroxylase which plays an important role in converting dietary vitamin D to active metabolite, 25-(OH)D3. We identified high levels of CYP2R1 expression using tissue microarray of human OSCC tumor specimens compared to normal adjacent tissue. Therefore, we hypothesize that 1,25(OH)₂D3 regulates CYP2R1 gene expression in OSCC tumor cells. Interestingly, real-time RT-PCR analysis of total RNA isolated from OSCC cells (SCC1, SCC11B, and SCC14a) treated with 1,25(OH)₂D3 showed a significant increase in CYP2R1 and vitamin D receptor (VDR) mRNA expression. Also, Western blot analysis demonstrated that 1,25(OH)₂D3 treatment time-dependently increased CYP2R1 expression in these cells. 1,25(OH)₂D3 stimulation of OSCC cells transiently transfected with the hCYP2R1 promoter (?2 kb)-luciferase reporter plasmid demonstrated a 4.3-fold increase in promoter activity. In addition, 1,25(OH)₂D3 significantly increased c-Fos, p-c-Jun expression, and c-Jun N-terminal kinase (JNK) activity in these cells. The JNK inhibitor suppresses 1,25(OH)₂D3, inducing CYP2R1 mRNA expression and gene promoter activity in OSCC cells. Furthermore, JNK inhibitor significantly decreased 1,25(OH)₂D3 inhibition of OSCC tumor cell proliferation. Taken together, our results suggest that AP-1 is a downstream effector of 1,25(OH)₂D3 signaling to modulate CYP2R1 gene expression in OSCC tumor cells, and vitamin D analogs could be potential therapeutic agents to control OSCC tumor progression.     

Induction of apoptosis in androgen-independent mouse mammary cell line by 1, 25-dihydroxyvitamin D3

Androgen-dependent tumors eventually progress to independent-tumors after androgen withdrawal. Effective treatment for hormone-independent tumors is therefore needed. Androgen-independent CS-2 cells could grow in serum-free culture whether androgen is present in the medium or not. In the present study, the mechanism of cell death in CS-2 cells was examined after 1, 25-dihydroxyvitamin D₃ [1, 25(OH)₂D₃] treatment. 1, 25(OH)₂D₃ has been examined as an anti-tumor agent, but its role in promoting cell death is poorly understood. Based upon the temporal sequence of DNA fragmentation, morphologic changes and loss of cell viability, the cells underwent apoptosis with 1, 25(OH)₂D₃treatment. Northern-blot analysis was used to identify a series of genes whose expression per cell is enhanced during the apoptotic pathway. In the apoptotic process induced by 1, 25(OH)₂D₃, mRNA expression of testosterone-repressed prostatic message 2, transforming growth factor β1, glucose-regulated 78-kDa protein and calmodulin increased. Flow-cytometric analysis showed that 1, 25(OH)₂D₃ treatment resulted in a block in G₀/G₁ of the cell cycle. These results demonstrate that androgen-independent CS-2 cells retain the ability to undergo apoptosis by 1, 25(OH)₂D₃. This system appears to be a good model for investigating apoptosis of hormone-independent cancer. © 1996 Wiley-Liss, Inc.     

DNA base modifications in renal chromatin of wistar rats treated with a renal carcinogen,ferric nitrilotriacetate

The C-myc gene encodes a nuclear protein whose precise function is still not folly understood. Introduction of a c-myc gene into a number of cell lines leads to an increase in their susceptibility to NK-cell lysis. It was reported earlier that c-myc can induce a decrease in the membrane expression of the MHC class-1 molecules and this may be one of the factors that render target cells relatively more susceptible to NK lysis. In this contribution, we show, in a human LCL line transfected with a constitutively active c-myc gene, an increased sensitivity to NK lysis, which correlates with an augmented effector-target binding ability of c-myc-transfected LCLs and with a high 1CAM-1 expression rather than with down-regulation of MHC class-l W6/32 epitope expression. © 1994 Wiley-Liss, Inc.     

Characterization of C57BL/6 plasmacytomas lacking a c-myc translocation

BALB/c peritoneal plasmacytomas induced by a variety of agents are invariably associated with a c-myc translocation. In contrast, naturally arising bone marrow plasma cell tumors in C57BL/KaLwRij mice lack this translocation. This difference has led to the suggestion that these are 2 fundamentally different plasma cell diseases. Herein, we have analyzed 2 rare C57BL/6 peritoneal plasmacytomas in terms of characteristics associated with the bone marrow–derived lines. Like the bone marrow lines, these peritoneal plasmacytomas do not exhibit c-myc translocations, indicating that c- myc translocation is not an obligatory event in the development of all murine extramedullary plasmacytomas. However, myc is dysregulated at the mRNA level, indicating that myc over-expression may be fundamental to most plasma cell diseases but that dysregulation can occur by alternative mechanisms possibly reflecting different genetic backgrounds. Int. J. Cancer 72:892–897, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.