Amplification of specific DNA sequences in C127 mouse cells transformed by bovine papillomavirus type 4

A rearranged bovine papillomavirus type 4 DNA fragment, present in a line of transformed C127 cells, was molecularly cloned into lambda GT 10. The rearranged viral fragment was found to consist of two separate sequences of 3.8 kb each. Clone A comprised 3.3 kb of host DNA linked to 0.5 kb of BPV-4 DNA. Clone B consisted of 2.2 kb of host DNA linked to 1.5 kb of BPV-4 DNA. The AB locus was found to be amplified and rearranged in a number of different BPV-4 transformed C127 cell lines, irrespective of the presence of viral DNA. The rearranged amplified locus was transferred and maintained in second and third round transformants. We propose that the altered AB locus is involved in the maintenance of the transformed phenotype in the absence of the BPV-4 DNA, and may act as an activated oncogene.     

Expression of a retinoic acid receptor gene in myeloid leukemia cells

Retinoic acid (RA) has been shown to increase differentiation in some leukemia cell lines (HL-60 and KG-1) but not others (K562). Similarly, RA has been reported to have variable effects on fresh blast cells. Recently, molecular clones have been obtained for the nuclear receptor for retinoic acid. The experiments described in this paper were designed to compare expression of the receptor to biological activity in myeloblastic leukemia cells. In four continuous AML cell lines, a positive correlation was found between retinoic acid receptor (RAR) expression by Northern analysis or RNA dot blot and the ability of RA to inhibit colony formation. Kinetic studies of the most sensitive cell line showed that inhibition of colony formation was associated with reduced blast cell self-renewal and differentiation-like events. RAR was detected in freshly obtained blast cells from 23 AML patients. Patient-to-patient variation was observed; however, a correlation was not found between RAR expression and response of the freshly obtained blast cells to RA.     

Benzoyl peroxide promotion of transformation of JB6 mouse epidermal cells: inhibition by ganglioside GT but not retinoic acid

Benzoyl peroxide (BzPo), a free radical generator with tumorpromoting activity on mouse skin, is shown to promote neoplastictransformation of JB6 mouse epidermal cells in vitro. Repeatedexposures to BzPo are required to readily detect promotion oftransformation of JB6 cells. Markedly reduced net synthesisof the major epidermal ganglioside, trisialoganglioside GT¹b,(G^T) occurs with BzPo treatment as with other tumor promotersactive in this system. Addition of ganglioside G^T prevents transformationby BzPo while retinoic acid does not.     

Nuclear but not mitochondrial genome involvement in 3-methylcholanthrene-induced expression of tumorigenicity in mouse somatic cells

The involvement of heritable modifications of mitochondrial DNA (mtDNA) in chemical carcinogenesis was examined by studies on the effects on tumorigenicity of interchange of mtDNA between 3-methylcholanthrene (MCA)-induced mouse tumor cells and nontumorigenic mouse cells by the cytoplast-to-cell fusion technique. The difference in propagating abilities of two types of mouse mtDNA, type 1 mtDNA of B10mtJ strain and type 2 mtDNA of C57BL/10 strain, was applied successfully for complete replacement of the host cell mtDNA by cytoplasmically transmitted mtDNA. Tumorigenicity was assayed in nude mice by inoculating 5 x 10(6) cells s.c. into the backs of the mice. The results showed that tumorigenicity was not induced in nontumorigenic cells by replacement of their mtDNA by that from MCA-induced tumor cells. Moreover, the tumorigenicity of MCA-induced tumor cells was still expressed when their mtDNA was replaced by that from normal cells with a limited life span. These observations suggest that, even if MCA treatment causes heritable modifications of mtDNA, modified mtDNA cannot induce chemical carcinogenesis and that modifications of nuclear DNA alone are sufficient for the expression of tumorigenicity.     

The modified firefly luciferase reporter gene (luc+) but not Renilla luciferase is induced by all-trans retinoic acid in MCF-7 breast cancer cells

Luciferase genes are widely used as reporters to analyze promoter and regulatory elements. We found that a luciferase reporter gene vector with a modified firefly luciferase gene (luc+), but not Renilla luciferase (Rluc), was induced by all-trans retinoic acid (tRA) in the MCF-7 breast cancer cell line. tRA (5 × 10^–6 M) increased luciferase activity of the pGL3 promoter vector (containing luc+) up to 3.8-fold in MCF-7 cells, but not in LNCaP prostate cancer cells or JEG-3 choriocarcinoma cells. Chimeric plasmids were constructed and showed that tRA-induction required the luc+ gene, but not any specific promoter or vector sequence. Time course and dose-response studies of tRA-induction indicated that longer treatment (>24 h) and higher tRA dose (>10^–6 M) were required for luc+ induction compared with those for a positive retinoic acid response element (maximum induction at 6 h and 10^–8 M tRA). Studies with the translation inhibitor, cycloheximide, indicated the half-life of the luc+ protein was increased from 9.7 ± 1.5 to 22.1 ± 3.1 h with tRA treatment. Other retinoids, TTNPB, a retinoic acid receptor /-specific ligand, and a retinoid X receptor ligand, did not significantly increase luc+ expression. Caution is needed in analysis of retinoid responsive gene regulation with the luciferase reporter system in MCF-7 cells, especially at high retinoid concentrations.     

A keratin epitope that is exposed in a subpopulation of preneoplastic and neoplastic mouse mammary epithelial cells but not in normal cells

Three monoclonal anti-keratin antibodies, AE1, AE3, and AE4, were used to compare the expression of keratins in normal, preneoplastic, and malignant mouse mammary epithelial cells growing in primary culture. In indirect immunofluorescence, AE1 did not stain normal cells but did stain a minority of preneoplastic and carcinoma cells. AE3 reacted with a subpopulation of epithelial cells in both the normal and abnormal cultures, except for certain cultures from one type of tumor wherein all of the epithelial cells were reactive. AE4 decorated an elaborate keratin filament network in all cultured mammary epithelial cells, regardless of neoplastic state. In double-label immunofluorescence, a guinea pig anti-keratin antiserum, which reacts preferentially with myoepithelial cells, exhibited coincident staining with AE1 in the tumor cultures and AE3 in the normal and most tumor cultures, indicating that the cells recognized by the antibodies in these populations were myoepithelial. Immunoblot experiments with cytoskeletal polypeptides extracted from the normal and tumor cells demonstrated that the set of keratins recognized by each monoclonal antibody was essentially the same in all of the cells except for a Mr 40,000 component that was present in normal cells but either absent or diminished in the cancer cells. Thus, while normal cells had Mr 40,000 and 50,000 keratins recognized by AE1, the epitope detected by this antibody was apparently concealed or "masked" in situ. AE3 reacted in immunoblots with a major keratin group (Mr 54,000-55,000) and a minor keratin (Mr 57,000), while AE4 reacted only with the Mr 54,000-55,000 keratin species. Because immunofluorescence with AE4 showed that the Mr 54,000-55,000 keratin group was present in all mammary epithelial cells, the AE3-reactive epitope must be masked in the majority of normal and tumor cells. The data therefore showed that epitopes on three major keratins, the Mr 40,000, 50,000, and 54,000-55,000 group, were "masked" in normal cells, whereas in tumor cells "masking" involved primarily the Mr 54,000-55,000 keratin. Attempts to "unmask" the epitopes recognized by AE1 in normal cells or to increase the number of cells reactive with AE3 in the normal and tumor cultures failed. Thus, certain cultured preneoplastic and neoplastic mammary cells with a myoepithelial phenotype have an altered organization of keratins that is manifested by a keratin antigenic determinant which is visible by immunocytochemistry in the abnormal cells but not in normal mouse mammary cells. This is the first demonstration that the immunoreactivity of keratins can be modified during neoplastic progression of epithelial cells.     

Expression of the human beta globin gene and 5'-deletion mutants in erythroleukemic mouse cells studied by DNA mediated gene transfer

A recombinant plasmid pTKH beta GHp-3 carrying genomic human DNA sequences coding for the beta globin gene covalently linked to the thymidine kinase gene of HSV-1 and the ampicillin resistance region of a bacterial plasmid has been constructed. Using Bal 31 exonuclease, deletion mutants have been obtained from a single HpaI site located 800 bp upstream the 5' end of the beta globin gene and towards the cap site. Recipient mouse erythroleukemic Friend TK- cells were transformed with these molecules. Analysis of donor DNA sequences in transformed cells by Southern blotting and filter hybridization has demonstrated the presence of full length multiple copies. RNA isolated from transformed non-induced Friend cells, carrying one or the other of the donor human beta globin deletion mutants, has been analysed by Northern blot and spot hybridization analyses. Evidence has been obtained which suggests that DNA sequences located upstream the CCAAT box (at -76 bp from the cap site) are required for optimal levels of human beta globin specific RNA in transformed cells.     

Expression of thymidylate synthase in human cells is an early G(1) event regulated by CDK4 and p16INK4A but not E2F

Thymidylate synthase (TS) is the enzyme that catalyses the last step in de novo thymidylate synthesis. It is of interest clinically because it is an effective target for drugs such as 5-fluorouracil, often used in combination therapy. Despite a number of earlier reports indicating that TS is a cell cycle-dependent enzyme, this remains equivocal. Here, we show that in HCT116 cells synchronised by serum starvation, there is a clear dissociation between the expression of cyclin E (a well-characterised cell-cycle protein) and TS. Although both cyclin E and TS mRNA and protein increased during G(1), TS upregulation was delayed. Moreover, TS levels did not decrease following S-phase completion while cyclin E decreased sharply. Similarly, clear differences were seen between cyclin E and TS as asynchronously growing HCT116 cells were growth-inhibited by low-serum treatment. In contrast to previous reports using rodent cells, adenovirus-mediated over-expression of E2F1 and cyclin E in three human cell lines had no effect on TS. Cell-cycle progression was blocked by treatment of cells with pharmacological inhibitors of CDK2 and CDK4 and by ectopic expression of p16INK4A. Whereas CDK2 inhibition had no effect on TS levels, inhibition of CDK4 was associated with decreased TS protein levels. These results provide the first evidence that drugs targeting CDK4 may be useful with anti-TS drugs as combination therapy for cancer.     

Induction of differentiation in HL-60 cells by retinoic acid and lymphocyte-derived differentiation-inducing factor but not by recombinant G-CSF and GM-CSF

Various concentrations of retinoic acid (RA, 10⁻⁹ to 10⁻⁷ M), lymphocyte-derived differentiation-inducing factor (DIF, 10–30%), and recombinant human G-CSF (100–4000 U/ml) and GM-CSF (100–4000 U/ml) were used to induce the differentiation of the HL-60 promyelocytic leukemia cells. Retinoic acid at a concentration of 10⁻⁷M could significantly inhibit the growth of HL-60 cells both in suspension and in soft agar cultures, and induced these cells to differentiate into mature granulocytes capable of reducing nitro-blue tetrazolium and ingesting latex beads. Thirty per cent (v/v) DIF was also an effective inducer of HL-60 cell differentiation, but it triggered the cells to mature into monocytes rather than granulocytes. In contrast, rG-CSF and rGM-CSF had no growth inhibitory effect on HL-60 cells either in suspension or in agar cultures at all concentrations tested, nor could these factors induce HL-60 cells to acquire the more mature granulocytic or monocytic phenotypes. Furthermore, rG-CSF/rGM-CSF had no differentiation-enhancing effect when added to RA-containing HL-60 cultures. These results argue against the efficacy of using CSFs for the treatment of myelocytic leukemia based on the principle of differentiation induction.     

P-glycoprotein is not expressed in a majority of colorectal carcinomas and is not regulated by mutant p53 in vivo.

Overexpression of the MDR1 product, P-glycoprotein (Pgp), has been shown to be one of the mechanisms underlying the development of multidrug resistance (MDR). Recently, one mutant p53 has been shown to stimulate the MDR1 gene promoter in vitro, whereas wild-type p53 repressed this activity. We measured Pgp and p53 expression by immunoblotting in 34 colorectal tumours, and performed mutation analyses on the p53-positive tumours to confirm the presence of mutant p53 protein. Tumour DNA indices (DIs) were also measured using flow cytometry. Pgp was detected in 44% (15/34) of the tumours and in 100% (13/13) of the normal mucosas (P = 0.0005), with highest levels of expression seen in normal mucosa, suggesting that initial drug resistance in colorectal tumours is not caused by Pgp. Highly DNA aneuploid tumours demonstrated the lowest levels of Pgp expression relative to moderately aneuploid and diploid colorectal tumours. p53 protein was detected in 53% (18/34) of the tumours, and 12 of 14 p53-positive tumours had p53 gene mutations, p53-negative tumours had approximately twice the level of Pgp expression of p53-positive tumours. Pgp expression was not associated with either p53 expression (P = 0.73) or incidence of p53 gene mutation (P = 0.70), suggesting that mutant p53 does not induce Pgp overexpression in colorectal carcinomas.     

Expression of the fibroblast growth factor 4 (FGF-4) gene is regulated by serum in Tera-2 embryonal carcinoma cells

Expression of the fibroblast growth factor 4 (FGF-4) gene is tightly regulated during mammalian development. Dysregulation of FGF-4 gene expression results in cell transformation and tumorigenesis. It is therefore pertinent to investigate the regulatory mechanisms which control expression of FGF-4. In an initial attempt to identify exogenous factors other than retinoic acid which might control FGF-4 expression, we have investigated the response of endogenous FGF-4 to serum in a number of embryonal carcinoma and embryonic stem cell lines. We have identified a human embryonal carcinoma cell line (Tera-2) in which the FGF-4 gene can be induced by serum. In Tera-2 cells made quiescent by serum deprivation, expression of the FGF-4 gene is repressed. Subsequent addition of serum reactivates FGF-LC expression and further addition of cycloheximide results in superinduction of mRNA suggesting that FGF-4 may be classified as an early response gene. It is suggested that this observation may be explained, at least in part, by the stage of differentiation of the Tera-2 cells.     

Expression of the NF-kappaB-responsive gene BTG2 is aberrantly regulated in breast cancer

BTG2, a p53-inducible antiproliferative gene, is stimulated in breast cancer cells by activation of nuclear factor kappa B (NF-kappaB). In rat mammary glands, BTG2 is expressed in epithelial cells and levels decreased during pregnancy and lactation but recovered during involution. Estrogen and progestin suppress BTG2 expression, suggesting that these steroids, which stimulate proliferation and lobuloalveolar development of mammary epithelial cells, may downregulate BTG2 in the mammary gland during pregnancy. Consistent with the report that BTG2 inhibits cyclin D1 expression, suppression of BTG2 mRNA in the mammary gland during gestation, and by estrogen and progestin, correlated with stimulation of cyclin D1. Ectopic expression of BTG2 inhibited breast cancer cell growth by arresting cells in the G1 phase, an effect reversed by cyclin D1. BTG2 expression was very low or undetectable in human breast cancer cell lines compared with nontumorigenic mammary epithelial cells, and nuclear expression of BTG2 was absent in 65% of human breast tumors compared with adjacent matched normal glands. Spontaneous mammary tumors arising in a mouse model with targeted expression of the early region of the SV40 large tumor Ag demonstrated loss of BTG2 protein very early during the tumorigenic process. Thus deregulation of BTG2 may be an important step in the development of mammary tumors.