Mouse Mammary Cancer Cell Line Research Articles

Tocotrienols induce apoptosis in breast cancer cell lines via an endoplasmic reticulum stress-dependent increase in extrinsic death receptor signaling

Tocotrienols are naturally occurring forms of vitamin E based on their structural similarity. This study focused on investigating anticancer effects of tocotrienols and the mechanisms of apoptosis induction by tocotrienols in vivo and in vitro. Dietary delivery of γ-tocotrienol (γ-T3) suppressed tumor growth in a syngeneic implantation mouse mammary cancer model by inhibiting cell proliferation and inducing apoptosis. In cell culture studies, γ-T3 inhibited colony formation of a mouse mammary cancer cell line and human breast cancer cell lines. The anti-proliferative effects of tocotrienols were highly correlated with an increase in apoptosis based on Annexin V assessment. Treatment of human MDA-MB-231 and MCF-7 cells with γ-T3 induced cleavages of PARP as well as caspase-8, -9, and -3. Additional analyses showed that γ-T3 activated c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK, and upregulated death receptor 5 (DR5) and C/EBP homologous protein (CHOP), an endoplasmic reticulum (ER) stress marker. Silencing either JNK or p38 MAPK reduced the increase in DR5 and CHOP and partially blocked γ-T3-induced apoptosis. Both DR5 and CHOP upregulation were required for γ-T3-induced apoptosis, and DR5 was transcriptionally regulated by CHOP after γ-T3 treatment. Moreover, γ-T3 increased the level of other ER-stress markers. Taken together, these results suggest that upregulation of DR5 by γ-T3 treatment is dependent on JNK and p38 MAPK activation which is mediated by ER-stress.

Ligand activation of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits cell growth in a mouse mammary gland cancer cell line

The effects of ligand activation of PPARβ/δ were examined in the mouse mammary tumor cell line (C20). Expression of PPARβ/δ was markedly lower in C20 cells as compared to the human non-tumorigenic mammary gland derived cell line (MCF10A) and mouse keratinocytes. Ligand activation of PPARβ/δ in C20 cells caused upregulation of the PPARβ/δ target gene angiopoietin-like 4 ( Angptl4). Inhibition of C20 cell proliferation and clonogenicity was observed following treatment with GW0742 or GW501516, two highly specific PPARβ/δ ligands. In addition, an increase in apoptosis was observed in C20 cells cultured with 10 μM GW501516 that preceded the observed inhibition of cell proliferation. Results from this study show that proliferation of the C20 mouse mammary gland cancer cell line is inhibited by ligand activation of PPARβ/δ due in part to increased apoptosis.

In vitrointeraction between mouse breast cancer cells and mouse mesenchymal stem cells during adipocyte differentiation

Surgical treatment following breast cancer, i.e. lumpectomy and mastectomy, may not efficiently remove all cancerous cells. As such, when mesenchymal stem cells (MSCs) are incorporated into the breast reconstruction process, it is likely that those MSCs will encounter remnant cancerous cells after transplantation into the defect site. The potential interaction between breast cancer cells and MSCs remains unclear. We hypothesized that paracrine interactions might occur between cells and various proteinases, growth factors and other cytokine molecules in the local microenvironment. Conditioned media (CM) from two mouse mammary cancer cell lines (4T1 and 4T07) and one mouse mammary epithelial cell line (NMuMG) were studied in the experimental model. Post-confluent mouse MSCs (D1 cells) were differentiated with an adipogenic hormonal cocktail. Conditioned media from the three cell types did not have an inhibitory effect on D1 cell viability; however, triglyceride (TG) and Oil red O (ORO) analysis results showed that 4T1-CM significantly inhibited D1 adipocyte differentiation and reduced lipid vesicle accumulation in the differentiating D1 cells. Preliminary analysis of the conditioned media revealed that a higher presence of matrix metalloprotease-9 (MMP-9) and urokinase plasminogen activator (uPA) was present in the 4T1-CM as compared to the levels found in 4T07-CM and NMuMG-CM, which were below the detection limit. Additionally, the conditioned medium of differentiated D1 cells on day 12 had a negative effect on 4T1 and 4T07 cell viability but no effect on NMuMG cell viability. The results suggest that mouse breast cancer cells modulate mouse MSC adipogenic differentiation, the level of modulation specific to the metastatic level.

Identification of the H2‐Kd‐restricted cytotoxic T lymphocyte epitopes of a tumor‐associated antigen, SPARC, which can stimulate antitumor immunity without causing autoimmune disease in mice

We previously reported that the secreted protein acidic and rich in cystein (SPARC) was overexpressed in melanoma in humans, and the serum SPARC level was useful as a novel tumor marker for melanoma. SPARC was also reported to be overexpressed in various human cancers. In this study, we asked whether SPARC-specific cytotoxic T lymphocytes (CTL) could induce antitumor immunity to SPARC-expressing tumor in mice or not as a preclinical study of SPARC-directed anticancer immunotherapy. Because of similarities in the structural motifs of major histocompatibility complex-binding peptides between H2-Kd and HLA-A24 (A*2402), the most common human leukocyte antigen class I allele in the Japanese population, we attempted to identify the H2-Kd-restricted SPARC epitope for CTL in BALB/c mice and we found that the mouse SPARC143-151 (DYIGPCKYI) and SPARC225-234 (MYIFPVHWQF) peptides could induce peptide-reactive CTL in BALB/c mice without causing autoimmune diseases. The immunization of mice with SPARC225-234 peptide-pulsed bone marrow-derived dendritic cells (BMDC) inhibited the growth of s.c. inoculated mouse mammary cancer cell line, N2C, expressing SPARC and these mice lived longer than the mice immunized with peptide-unpulsed BMDC. In conclusion, our study indicated that SPARC peptide-based cancer immunotherapy was effective and safe at least in a mouse tumor prevention model.

Increased risk of cognitive impairment or dementia in women who underwent oophorectomy before menopause

There is increasing laboratory evidence for a neuroprotective effect of estrogen; however, the clinical and epidemiologic evidence remains limited and conflicting. We studied the association of oophorectomy performed before the onset of menopause with the risk of subsequent cognitive impairment or dementia. We included all women who underwent unilateral or bilateral oophorectomy before the onset of menopause for a non-cancer indication while residing in Olmsted County, MN, from 1950 through 1987. Each member of the oophorectomy cohort was matched by age to a referent woman from the same population who had not undergone oophorectomy. In total, we studied 813 women with unilateral oophorectomy, 676 women with bilateral oophorectomy, and 1,472 referent women. Women were followed through death or end of study using either direct or proxy interviews. Women who underwent either unilateral or bilateral oophorectomy before the onset of menopause had an increased risk of cognitive impairment or dementia compared to referent women (hazard ratio [HR] = 1.46; 95% CI 1.13 to 1.90; adjusted for education, type of interview, and history of depression). The risk increased with younger age at oophorectomy (test for linear trend; adjusted p < 0.0001). These associations were similar regardless of the indication for the oophorectomy, and for women who underwent unilateral or bilateral oophorectomy considered separately. Both unilateral and bilateral oophorectomy preceding the onset of menopause are associated with an increased risk of cognitive impairment or dementia. The effect is age-dependent and suggests a critical age window for neuroprotection.

Anti-proliferative and mutagenic activities of aqueous and methanol extracts of leaves from Pereskia bleo (Kunth) DC (Cactaceae)

The anti-proliferative effects of the aqueous and methanol extracts of leaves of Pereskia bleo (Kunth) DC (Cactaceae) against a mouse mammary cancer cell line (4T1) and a normal mouse fibroblast cell line (NIH/3T3) were evaluated under an optimal (in culture medium containing 10% foetal bovine serum (FBS)) and a sub-optimal (in culture medium containing 0.5% FBS) conditions. Under the optimal condition, the aqueous extract showed a significant ( p < 0.05) anti-proliferative effect at 200 μg/mL and 300 μg/mL in 4T1 cells and 300 μg/mL in NIH/3T3 cells, whereas the methanol extract did not show any notable anti-proliferative effect in these cell lines, at any of the concentrations tested. Under the sub-optimal condition, the aqueous extract showed a significant ( p < 0.05) anti-proliferative effect at 200 μg/mL and 300 μg/mL in NIH/3T3 cells, whilst the methanol extract showed a significant ( p < 0.05) anti-proliferative effect at 200 μg/mL and 300 μg/mL in both cell lines. An upward trend of apoptosis was observed in both 4T1 and NIH/3T3 cells treated with increasing concentrations of the aqueous extract. The level of apoptosis observed at all the concentrations of the aqueous extract tested was consistently higher than necrosis. There was a significant ( p < 0.05) increase in the level of necrosis observed in the 4T1 cells treated with 300 μg/mL of the methanol extract. Generally, the level of necrosis was noted to be higher than that of apoptosis in the methanol extract-treated cells. The mutagenicity assay performed showed that in the absence of S-9 liver metabolic activation, the extract was not mutagenic up to the concentration of 165 μg/mL. However, in the presence of S-9 liver metabolic activation, the aqueous extract was mutagenic at all the concentrations tested. This study shows that both the aqueous and methanol extracts of the leaves from Pereskia bleo (Kunth) DC (Cactaceae) do not have appreciable anti-proliferative effect on the 4T1 and NIH/3T3 cells as the EC 50 values obtained are greater than 50 μg/mL when tested under optimal culture condition. Moreover, the aqueous extract may form mutagenic compound(s) upon the metabolisation by liver enzymes.

Contrast-enhanced in vivo imaging of breast and prostate cancer cells by MRI: Rodriguez O, Fricke S, Chien C, Dettin L, VanMeter J, Shapiro E, Dai HN, Casimiro M, Ileva L, Dagata J, Johnson MD, Lisanti MP, Koretsky A, Albanese C, Lombardi Cancer Center, Department of Oncology, Georgetown University Medical Center, Washington, DC

The development of effective cancer therapies has been hampered, in part, by the inability to noninvasively follow tumor progression from the initial cancerous lesion through to metastasis. We have previously shown that superparamagnetic iron oxide particles can be used as magnetic resonance imaging contrast agents to label embryonic, mesenchymal, and hematopoietic stem cells in vivo. Improving the capacity to noninvasively image cancer progression is an appealing method that could be useful for assessing the efficacy of anticancer therapies. We have established that human prostate (LNCaP, DU145, PC3), rodent prostate (TRAMPC1, YPEN-1), human breast (MDA-MB-231), and mouse mammary (Myc/VEGF) cancer cell lines were readily labeled by fluorescent superparamagnetic submicron particles of iron oxide (MPIOs). The MPIOs were essentially inert with respect to cell proliferation and tumor formation. Fluorescence stereomicroscopy and three dimensional magnetic resonance imaging (MRI) determined that subcutaneous, intramuscular, or orthotopically implanted labeled cancer cells could be imaged, in vivo, despite in some cases being undetectable by manual palpation. The MPIO-labeled cancer cells could also be imaged, in vivo, at least 6 weeks after implantation. The fluorescent MPIOs further allowed for the ex vivo identification of tumors cells from histological sections. This study demonstrates the feasibility of using fluorescent MPIOs in prostate and breast cancer cell lines as both a negative contrast agent for in vivo MRI as well as a fluorescent tumor marker for optical imaging in vivo and ex vivo.

Involvement of hepatocyte growth factor in the development of bone metastasis of a mouse mammary cancer cell line, BALB/c-MC

Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of certain cancer cells. After tumors metastasize to bone, they stimulate osteoclastogenesis and expand in the bone tissue. Hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for hepatocytes, promotes tumor growth, invasion and metastasis. HGF is mainly produced by cells of mesenchymal origin, and osteoblasts/osteocytes and bone marrow stromal cells originate from mesenchymal cells. However, it is not clear what effect HGF has on tumor progression in bone metastasis. In the present study, we investigated the roles of HGF in bone metastasis using the mouse mammary cancer cell line BALB/c-MC. Cancer cells injected into hearts of mice metastasized to bone in their hind limbs. HGF immunoreactivity was detected in the stroma surrounding the tumor nests, and blood vessels expressing CD31 (a marker of endothelial cells) were observed in the HGF-positive area. To identify the cells producing HGF, we measured concentration of HGF in culture media. HGF concentration was elevated in osteoblast cultures (3.13 ± 0.25 ng/ml), whereas HGF was undetectable (<0.4 ng/ml) in BALB/c-MC and bone marrow cell cultures. HGF concentration in osteoblast cultures increased 2.5-fold in response to 10 −6 M PGE 2. Addition of HGF to BALB/c-MC cultures caused doubling of the cell number. Moreover, Western blot analysis revealed expression of c-Met/HGF receptor by BALB/c-MC. In the Matrigel invasion chamber assay, addition of HGF to the bottom well increased the rate at which BALB/c-MC invaded the bottom well through the membrane. Furthermore, when osteoblasts were cultured in the bottom well, the number of BALB/c-MC cells that invaded the bottom well through the membrane increased 3.7-fold, compared to assays without osteoblasts. Addition of NK4, an inhibitor of HGF, completely abolished the enhancement of the invasive potential of the BALB/c-MC cells in the presence of osteoblasts. These findings suggest that HGF produced by osteoblasts induces migration of cancer cells from sinusoidal capillaries to bone marrow space and stimulates growth of cancer cells in the bone microenvironment. Thus, osteoblasts appear to promote bone metastasis of some cancers via HGF-c-Met signaling.

Contrast-Enhanced In Vivo Imaging of Breast and Prostate Cancer Cells by MRI

The development of effective cancer therapies has been hampered, in part, by the inability to non-invasively follow tumor progression from the initial cancerous lesion through to metastasis. We have previously shown that superparamagnetic iron oxide particles can be used as magnetic resonance imaging contrast agents to label embryonic, mesenchymal and hematopoietic stem cells in vivo. Improving the capacity to non-invasively image cancer progression is an appealing method that could be useful for assessing the efficacy of anticancer therapies. We have established that human prostate (LNCaP, DU145, PC3), rodent prostate (TRAMPC1, YPEN-1), human breast (MDA-MB-213) and mouse mammary (Myc/VEGF) cancer cell lines were readily labeled by fluorescent superparamagnetic sub-micron particles of iron oxide (MPIOs). The MPIOs were essentially inert with respect to cell proliferation and tumor formation. Fluorescence stereomicroscopy and three dimensional magnetic resonance imaging (MRI) determined that subcutaneous, intramuscular or orthotopically implanted labeled cancer cells could be imaged, in vivo, despite in some cases being undetectable by manual palpation. The MPIO-labeled cancer cells could also be imaged, in vivo, at least 6 weeks after implantation. The fluorescent MPIOs further allowed for the ex vivo identification of tumors cells from histological sections. This study demonstrates the feasibility of using fluorescent MPIOs in prostate and breast cancer cell lines as both a negative contrast agent for in vivo MRI as well as a fluorescent tumor marker for optical imaging in vivo and ex vivo.

Preferential targeting of vesicular stomatitis virus to breast cancer cells

Vesicular stomatitis virus (VSV) is a candidate for development for cancer therapy. We created a recombinant replicating VSV (rrVSV) with an altered surface protein that targeted preferentially to breast cancer cells. The rrVSV genome contained a single glycoprotein (gp) gene derived from Sindbis virus. This gene expressed a chimeric Sindbis E2 binding gp and the native Sindbis E1 fusion gp. The chimeric E2 binding gp, called Sindbis-SCA-erbb2, was modified to reduce its native binding function and to contain a single chain antibody (SCA) with specificity for the human epidermal growth factor receptor Her2/neu protein, erbb2. These viruses selectively infected, replicated in and killed cells expressing erbb2. The titer of rrVSV on SKBR3 cells, a human breast cancer cell line which highly expresses erbb2 was 3.1 × 10 7/ml compared with a titer of 7.3 × 10 5/ml on 143 cells, a human osteosarcoma cell line which does not express erbb2. The titer of rrVSV on D2F2/E2 cells, a mouse mammary cancer cell line stably transfected to express human erbb2 was 2.46 × 10 6/ml compared with a titer of 5 × 10 4/ml on the parent D2F2 cells which do not express erbb2. When titered on erbb2-negative cells, non-replicating pseudotype VSV coated with Sindbis-SCA-erbb2 had <3% the titer of pseudotype VSV coated with wild type Sindbis gp indicating that the chimeric Sindbis gp had severely impaired binding to the natural receptor. Analysis of the protein composition of the rrVSV found low expression of the modified Sindbis gp on the virus.

Casein kinase II-mediated phosphorylation of NF-kappaB p65 subunit enhances inducible nitric-oxide synthase gene transcription in vivo.

Nitric oxide (NO) produced by inducible nitric-oxide synthase (NOSII) is mainly regulated at the transcriptional level by the nuclear factor-kappaB (NF-kappaB). In the present study, we further analyzed the role of NF-kappaB in the in vivo transcriptional regulation of NOSII gene by comparing two clones isolated from the EMT-6 mouse mammary cancer cell line. In response to interleukin (IL)-1beta or lipopolysaccharide (LPS), EMT-6 clone J (EMT-6J) cells produce 3-fold more NO than EMT-6 clone H (EMT-6H) cells, an effect correlated with enhanced activation of NF-kappaB in EMT-6J cells. In response to IL-1beta, the kinetics of degradation of NF-kappaB inhibitors IkappaB-alpha and IkappaB-beta, the nucleo-cytoplasmic shuttling of the transcription factor and its binding to a specific DNA sequence were similar in both clones. In contrast, an IL-1beta-induced phosphorylation of serine residues in NF-kappaB p65 subunit was observed in EMT-6J, but not in EMT-6H, cells. This IL-1beta-induced phosphorylation of p65 was specifically prevented by pretreatment of EMT-6J cells with the casein kinase II inhibitor DRB. Small interfering RNA-mediated depletion of casein kinase II-alpha subunit also decreased NF-kappaB transcriptional activity and NOSII gene transcription in IL-1beta and LPS-stimulated EMT-6J cells to the levels observed in EMT-6H cells treated in the same conditions. Altogether, these data indicate that casein kinase II-mediated phosphorylation of p65 subunit can enhance the transcriptional activity of NF-kappaB in vivo. This post-translational modification of the transcription factor can be responsible for increased NOSII gene transcription and NO production in tumor cells exposed to either IL-1beta or LPS.

Establishment of two hormone-responsive mouse mammary carcinoma cell lines derived from a metastatic mammary tumor.

We report the establishment of two mouse mammary cancer cell lines, MC7-2A and MC7-2B obtained from a mouse mammary carcinoma induced by medroxyprogesterone acetate (MPA) and maintained by syngeneic transplantation in BALB/c mice. They are epithelial (express cytokeratins) and express both estrogen receptors alpha (ERalpha) and progesterone receptors (PRs) isoforms A and B (western blots). In vitro, MPA inhibited 3H-thymidine uptake, starting from concentrations as low as 10(-13) M in MC7-2A and 10(10) M in MC7-2B; the antiprogestin RU 486 exerted a stimulatory effect at 10(-14) M in both cell lines; 17-beta-estradiol (E2) also exerted a stimulatory effect starting at 10(-10) M in MC7-2A and at 10(-13) M in MC7-2B. When transplanted in syngeneic mice, both cell lines originated adenocarcinomas that gave rise to lung metastases within 3 months. In in vivo studies, in MC7-2A, the antiprogestin inhibited completely tumor growth, E2 induced a slight although significant ( p < 0.05) stimulatory effect and MPA stimulated tumor growth while MC7-2B cells were unresponsive to all treatments. ER and PR were also expressed in tumors as assessed by immunohistochemistry. Two marker chromosomes were identified by FISH as translocations between chromosomes 4 and 7, and between chromosomes X and 2; the third marker chromosome remains unidentified. All these markers were also present in the parental tumor. A new marker, a centric fusion of chromosomes 2, was acquired in both cell lines. Considering that there are very few murine breast carcinoma responsive cell lines, these cells represent new tools in which the regulatory effect of hormones can be studied.

The effect of deletion of cyclooxygenase-2, prostaglandin receptor EP2, or EP4 in bone marrow cells on osteoclasts induced by mouse mammary cancer cell lines

The inducible prostaglandin (PG) synthesis enzyme, cyclooxygenase-2 (COX-2), is involved in osteoclast (OC) formation in cocultures of mouse mammary cancer cell lines (MMT060562 or BALB/c-MC) and bone marrow cells through production of PGE 2. There are four PGE 2 receptors but only the EP2 and EP4 receptors are reported to be important for OC formation. We have investigated the role of COX-2, EP2 receptor, and EP4 receptor in marrow cells for osteoclastogenesis in cocultures of cancer cells and bone marrow cells. We cocultured cancer cell lines with bone marrow cells from COX-2 knockout (−/−), EP2 −/− or EP4 −/− mice compared to wild-type mice. In addition, an EP4 receptor antagonist (EP4 RA) was added in some cocultures. Disruption of COX-2 gene in bone marrow cells had no effect on PGE 2 production and OC formation in cocultures with MMT060562, while it abrogated PGE 2 production and OC formation in cocultures with BALB/c-MC. Disruption of the EP2 gene in bone marrow cells had no effect on OC formation in the cocultures, while disruption of the EP4 gene in bone marrow cells abrogated OC formation in the cocultures. Furthermore, EP4 RA suppressed OC formation and prevented the increase in receptor activator of nuclear factor κB ligand (RANKL) mRNA levels in the cocultures. We conclude that COX-2 in cancer cells is responsible for PGE 2 and OC production in cocultures with MMT060562, while COX-2 in bone marrow cells, not cancer cells, is responsible for PGE 2 and OC production in cocultures with BALB/c-MC, and EP4 receptors are essential for OC formation in both cocultures.

Eradication of breast cancer xenografts by hyperthermic suicide gene therapy under the control of the heat shock protein promoter.

To investigate the usefulness of heat shock protein (HSP) promoter for breast cancer gene therapy, hyperthermia and HSV thymidine kinase (tk) suicide gene combination therapy was examined with mouse mammary cancer cell line FM3A. HSP promoter activity was markedly increased after heat shock (41-45 degrees C), with maximum activation (about 400-fold) at 3 hr. An in vitro cytotoxic assay showed that HSP-tk-transduced FM3A cells became more sensitive (more than 50,000 times) to ganciclovir (GCV) with heat shock, but untreated cells showed no increased cytotoxic sensitivity to GCV compared with control FM3A cells. In addition to promoter-oriented selective cell killing, a "chemosensitization effect" as a bystander effect was demonstrated by hyperthermia and suicide gene combination therapy, using a non-heat-inducible promoter. Immunohistochemical analysis revealed that this synergistic killing effect was dependent on apoptotic cell death with upregulation of both Fas and FasL (Fas ligand) expression. We also examined the efficacy of HSP-tk gene therapy in vivo by implanting breast cancer in subcutaneous and intraperitoneal models of BALB/c nude mice targeted by the HVJ-anionic liposome method. Significant tumor regression was observed in HSP-tk-transduced tumors followed by hyperthermia therapy, but no such inhibition was noted in either the mock vector transfection or hyperthermia group compared with control tumor-bearing mice. Our results demonstrate that this combination system is synergistically effective in mediating Fas-dependent apoptosis for a specific gene therapy targeting HSP-expressing mammary carcinomas, even in advanced and heat-resistant breast cancer.

Syntheses, antitumor activities, and antiangiogenesis ofexo-3,6-epoxy-1,2,3,6-tetrahydrophthalimidoethanoyl- 5-fluorouracil and its polymers

A new monomer, exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimidoethanoyl-5-fluorouracil (ETFU), was synthesized by the reaction of exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimidoethanoyl chloride (ETPC) and 5-fluorouracil (5-FU). The homopolymer of ETFU and its copolymers with acrylic acid (AA) and vinyl acetate (VAc) were prepared via photopolymerizations with 2,2-dimethoxy-2-phenylacetophenone at 25 °C for 48 h. The structures of the synthesized monomer and polymers were identified by Fourier transform infrared, 1H NMR, and 13C NMR spectroscopy and elemental analysis. The ETFU contents in poly(ETFU-co-AA) and poly(ETFU-co-VAc) were 26 mol % and 26 mol %, respectively. The number-average molecular weights of the polymers, as determined by gel permeation chromatography, ranged from 5600 to 17,000. The in vitro cytotoxicities of 5-FU and the synthesized samples against mouse mammary carcinoma and human histiocytic lymphoma cancer cell lines increased in the following order: ETFU > 5-FU > poly(ETFU-co-AA) > poly(ETFU) > poly(ETFU-co-VAc). The in vivo antitumor activities of the polymers against Balb/C mice bearing the sarcoma 180 tumor cells were greater than those of 5-FU at all doses tested. The inhibitions of the samples for SV40 DNA replication and antiangiogenesis were much greater than the inhibition of the control. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 4272–4281, 2000

Control of metastasis by Asn-linked, beta1-6 branched oligosaccharides in mouse mammary cancer cells.

Studies in cell lines and malignant human tissues have shown that increased cell-surface Asn-linked beta1-6(GlcNAcbeta1-6Man) branching is associated with increased tumorigenic and metastatic properties. In this study, three mouse mammary cancer cell lines were transfected with an expression vector containing the mouse cDNA for N-acetylglucosaminyltransferase V (GlcNAcT-V EC 2.4.1.155), the glycosyltransferase responsible for initiating beta1-6 branching on Asn-linked carbohydrates. The cell lines were screened for increased cytotoxicity to L-PHA, a lectin specific for beta1-6 branching structures. Cell lines exhibiting increased L-PHA cytotoxicity expressed increased levels of beta1-6 branching structures. Northern blots detected the presence of GlcNAcT-V transcribed from the expression vector in the L-PHA sensitive cell lines. After injection into the tail veins of mice, transfected cell lines with increased beta1-6 branching on the cell surface formed elevated levels of lung tumors relative to control transfected cell lines (P < 0.002). Western blots of membrane proteins from GlcNAcT-V transfected and control cells probed with the lectins DSA and WGA did not show an increase in polyN-acetyllactosamine and sialic acid content in the transfected cell lines. These results demonstrate that a specific increase in beta1-6 branching due to an elevation in GlcNAcT-V expression increases metastatic potential.

Antitumor activity of three mouse mammary cancer cell lines after interferon-gamma gene transfection.

The antitumor effects of three mouse mammary tumors transfected to express interferon-gamma were evaluated. Three immunologically different tumors were used: DA3, EMT6, and 410. All three cell lines were successfully transfected with highly efficient viral vectors. Wild type or transfected tumor cells were injected subcutaneously into Balb/c mice. Animals were observed for tumor growth and the induction of immunologic memory. A significant decrease occurred in the size of all transfected tumors, EMT6 1.9 cm2, DA3 1.7, and 410 1.8 compared with nontransfected control tumors with a mean size of 4 cm2 on day 30. To further test the development of immunity, animals were injected with either nontransfected or transfected tumors and challenged with nontransfected tumor. Animals immunized with transfected tumor cells had significantly smaller tumors, EMT6 2.5 cm2, DA3 3.1, and 410 2.4 compared with controls with a mean size of 4 cm2. No specific splenocyte cytotoxicity was shown. Expression of major histocompatibility complex class I antigens was enhanced in the 410 and DA3 tumor lines. Significant antitumor effects were observed after interferon-gamma gene transfection of three mouse mammary cancer cell lines. Up-regulation of major histocompatibility complex class I antigen expression is a partial explanation of these findings. These results provide preliminary studies for gene therapy of human breast cancer.

Inhibitory action of androstenedione on the proliferation and cell cycle kinetics of aromatase-free MXT and MCF-7 mammary tumour cell lines

The effects of androstenedione (AD) on cell proliferation and kinetics have been measured in MXT mouse and MCF-7 human mammary cancer cell lines using SAMBA 200 cell image analysis of Feulgen-stained nuclei. At a concentration of 0.01 μM AD inhibited the proliferation of both cell lines whereas a higher dose (1 μM) was inhibitory on MCF-7 cell proliferation but stimulatory in MXT cells. It is unlikely that these effects are due to aromization of AD into oestrogen since (a) both cell lines were devoid of aromatase and (b) both cell lines were similarly affected by oestradiol (E 2), being stimulated at low concentrations and inhibited at high doses. Furthermore, inhibition by AD seems to occur, at least in part, by blockade of the cell cycle whereas that by E 2 appears to be cell cycle independent.

Influence of fetal calf serum in combination with pharmacological doses of progesterone or estradiol on proliferation and cell cycle kinetics of cultured mammary cancer cells.

We describe an original method to monitor clonal cell density (hyperplasia) and the cell cycle kinetics of neoplastic cells simultaneously. We thus characterize the in vitro influence of two different types of fetal calf serum (FCS), defined as FCS-S and FCS-I, on the progesterone- or estradiol-induced effect on proliferation and cell cycle kinetic parameters of the MXT mouse and MCF-7 human mammary cancer cell lines. The two sera were treated with dextran-coated charcoal. The FCS-S serum showed a stimulatory influence on MXT and MCF-7 growth, whereas FCS-I was devoid of any clear-cut influence. The cells were cultured on glass coverslips, placed in Petri dishes containing either a control or a hormone-added medium, fixed for histology and submitted to the Feulgen reaction, which allows selective and quantitative (stoichiometric) staining of DNA. Proliferation and cell cycle kinetics were analyzed on the same sample of cells by means of the SAMBA 200 cell image processor. Our results show that steroid-mediated effects were dramatically modulated according to the type of serum used. Furthermore, they also show that pharmacological doses of progesterone or estradiol decrease MXT cell growth by at least two different mechanisms: the first is related to cell cycle kinetics, i.e. an inhibition of the cells into the S phase, while the second remains unknown but seems to be cell cycle independent. High dose estradiol, but not progesterone, induced the same inhibitory influence on the MCF-7 cells.

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging. The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6°C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to sialic acid-associated cell surface charges. The pattern of change in relation to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maximum occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.