Mammary Tumor-derived Cell Lines Research Articles

Abstract P5-01-10: A panel of murine mammary tumor cell lines to study immune responses to epitopes created by different mutagens including human APOBEC3 enzymes

Abstract Background: Tumor mutation loads are a strong predictor of immunotherapy benefit. This is evidenced by studies on skin, lung, and colon cancer, where well-defined processes elicit high mutation burdens and checkpoint therapies often provoke strong anti-tumor immune responses. In comparison, only a small number of breast cancer patients have responded to checkpoint therapies such as anti-PD-L1 and anti-PD-1 monoclonal antibodies. Approximately one-fourth of primary breast cancer and one-third of metastatic tumors have a significant APOBEC mutation burden defined as C-to-T and C-to-G mutations in TCA or TCT trinucleotide motifs, and many tumors have >10,000 of these APOBEC signature single base substitution mutations. However, to our knowledge, systematic immunotherapy studies focusing on this unique, hypermutated subset of breast cancers have yet to be done. Methods: To address the potential relationship between different checkpoint therapies and APOBEC mutation burden, we created a panel of >30 mammary tumor cell lines derived from fully immune-proficient C57BL/6 animals. At least 6 of these cell lines are immortalized, readily engineered using standard molecular biology procedures, and capable of re-engraftment and tumor formation in immune proficient C57BL/6 animals. Two lines were engineered to inducibly express human APOBEC3A, subjected to 1- to 20-rounds of sublethal mutagenesis, and tested systematically for checkpoint therapy responsiveness. Results: Two cell lines were engineered to inducibly express APOBEC3A or a catalytic mutant (E72A). Clones were characterized for inducible APOBEC3A expression and associated DNA damage responses. After varying rounds of mutagenesis, clones were engrafted into the subcutaneous flank or mammary fat pad of C57BL/6 animals. Immunohistochemical and flow cytometry analysis revealed a robust intratumoral CD8 T cell population including a subset with PD-1 expression. Additionally, engrafted tumor cells expressed high levels PD-L1. Experiments are ongoing and immunotherapy responses will be presented. Conclusions: We have isolated a panel of C57BL/6 mammary tumor-derived cell lines and demonstrated re-engraftment into immunocompetent animals. These cell lines constitute a platform for studying the relationship between different mutagens including APOBEC and immunotherapy responses. Citation Format: Jordan Naumann, Michael Carpenter, Prokopios Argyris, William Brown, Reuben Harris. A panel of murine mammary tumor cell lines to study immune responses to epitopes created by different mutagens including human APOBEC3 enzymes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-10.

Metabolomic profiling of mouse mammary tumor-derived cell lines reveals targeted therapy options for cancer subtypes.

Breast cancer is a heterogeneous disease with several subtypes that currently do not have targeted therapeutic options. Metabolomics has the potential to uncover novel targeted treatment strategies by identifying metabolic pathways required for cancer cells to survive and proliferate. The metabolic profiles of two histologically distinct breast cancer subtypes from a MMTV-Myc mouse model, epithelial-mesenchymal-transition (EMT) and papillary, were investigated using mass spectrometry-based metabolomics methods. Based on metabolic profiles, drugs most likely to be effective against each subtype were selected and tested. We found that the EMT and papillary subtypes display different metabolic preferences. Compared to the papillary subtype, the EMT subtype exhibited increased glutathione and TCA cycle metabolism, while the papillary subtype exhibited increased nucleotide biosynthesis compared to the EMT subtype. Targeting these distinct metabolic pathways effectively inhibited cancer cell proliferation in a subtype-specific manner. Our results demonstrate the feasibility of metabolic profiling to develop novel personalized therapy strategies for different subtypes of breast cancer. Schematic overview of the experimental design for drug selection based on breast cancer subtype-specific metabolism. The epithelial mesenchymal transition (EMT) and papillary tumors are histologically distinct mouse mammary tumor subtypes from the MMTV-Myc mouse model. Cell lines derived from tumors can be used to determine metabolic pathways that can be used to select drug candidates for each subtype.

A three-dimensional co-culture system to investigate macrophage-dependent tumor cell invasion.

Macrophages infiltrate cancers and promote progression to invasion and metastasis. To directly examine tumor-associated macrophages (TAMs) and tumor cells interacting and co-migrating in a three-dimensional (3D) environment, we have developed a co-culture model that uses a PyVmT mouse mammary tumor-derived cell line and mouse bone marrow-derived macrophages (BMM). The Py8119 cell line was cloned from a spontaneous mammary tumor in a Tg(MMTV:LTR-PyVmT) C57Bl/6 mouse and these cells form 3-dimensional (3D) spheroids under conditions of low adhesion. Co-cultured BMM infiltrate the Py8119 mammospheres and embedding of the infiltrated mammospheres in Matrigel leads to subsequent invasion of both cell types into the surrounding matrix. This physiologically relevant co-culture model enables examination of two critical steps in the promotion of invasion and metastasis by BMM: 1) macrophage infiltration into the mammosphere and, 2) subsequent invasion of macrophages and tumor cells into the matrix. Our methodology allows for quantification of BMM infiltration rates into Py8119 mammospheres and demonstrates that subsequent tumor cell invasion is dependent upon the presence of infiltrated macrophages. This method is also effective for screening macrophage motility inhibitors. Thus, we have developed a robust 3D in vitro co-culture assay that demonstrates a central role for macrophage motility in the promotion of tumor cell invasion.

The calcineurin/NFAT pathway is activated in diagnostic breast cancer cases and is essential to survival and metastasis of mammary cancer cells.

Nuclear factor of activated T cells 1 (NFAT1) expression has been associated with increased migratory/invasive properties of mammary tumor-derived cell lines in vitro. It is unknown, however, if NFAT activation actually occurs in breast cancer cases and whether the calcineurin/NFAT pathway is important to mammary tumorigenesis. Using a cohort of 321 diagnostic cases of the major subgroup of breast cancer, we found Cn/NFAT pathway activated in ER−PR−HER2− triple-negative breast cancer subtype, whereas its prevalence is less in other subgroups. Using a small hairpin RNA-based gene expression silencing approach in murine mammary tumor cell line (4T1), we show that not only NFAT1 but also NFAT2 and their upstream activator Cn are essential to the migratory and invasive properties of mammary tumor cells. We also demonstrate that Cn, NFAT1 and NFAT2 are essential to the tumorigenic and metastatic properties of these cells in mice, a phenotype which coincides with increased apoptosis in vivo. Finally, global gene expression analyses identified several NFAT-deregulated genes, many of them being previously associated with mammary tumorigenesis. In particular, we identified the gene encoding a disintegrin and metalloproteinase with thrombonspondin motifs 1, as being a potential direct target of NFAT1. Thus, our results show that the Cn/NFAT pathway is activated in diagnostic cases of breast cancers and is essential to the tumorigenic and metastatic potential of mammary tumor cell line. These results suggest that pharmacological inhibition of the Cn/NFAT pathway at different levels could be of therapeutical interest for breast cancer patients.

Abstract 555: Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line.

Abstract Retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through interactions with specific Protein Kinase C (PKC) isoforms. In this work we studied: A)- the modulation of PKCα and PKCδ expression when cells were treated with ATRA (1 μM), B)- The expression profile of Retinoic Acid Receptors (RAR) when cells are treated with ATRA and/or PKCα / PKC δ inhibitors (Gö6976 2.5 μM / Rottlerin 1 μM) and C)- the effect of combining ATRA and PKC inhibitors on in vitro cell growth, cell cycle distribution and senescence. We used as model the murine mammary tumor-derived cell line LM38-LP, composed by luminal (LEP) and myoepithelial (MEP) cells. By RT-PCR we could determine that 48 h ATRA treatment increased PKCδ only in the LM38-LP LEP population and decreased PKCα expression levels in both cellular components. Regarding RARs profile, the treatment with PKCα inhibitor Gö6976 reduced RARγ2 levels, while its combination with ATRA also increased RARβ2. Furthermore, 96 h treatment with ATRA, Gö6976, or Rottlerin or their combinations induced cell growth inhibition (cell number x105: control 22± 1.1; ATRA 11±0.1; Gö6976 3.6±0.6; Rottlerin 9±0.1; ATRA/Gö6976 2.3±0.03; ATRA/Rottlerin 6.7±1.3, p ≤0.05 vs control in all treatments). By flow cytometry we could determine that both ATRA and Rottlerin treatments induce LM38-LP cell cycle arrest in G1 phase, while Gö6976 increased the number of cells in the subG1 phase, correlating with increased apoptosis and confirmed by Annexin V-FITC staining. Employing β-galactosidase activity assay and bright field microscopy, we determined that 96 h ATRA treatment induced senescence mainly in the MEP component while Gö6976 does it in the LEP population. Interestingly, Rottlerin treated cells showed morphological alterations compatible with autophagy. We conclude that cell growth inhibition exerted by retinoids may be enhanced by the reduction in PKCα expression and / or activity. In addition the combination of ATRA plus Gö6976, induced differential responses on the different LM38-LP cell components, showing senescence in MEP cells, together with cell cycle arrest and apoptosis mainly in the LEP compartment. Consistently, all these biological effects correlated with an increase in RARβ2 (known to induce cell differentiation) and a decrease in RARγ2 (implicated in proliferation) mRNAs level. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 555. doi:10.1158/1538-7445.AM2013-555

Abstract P4-08-04: Abcc10 status affects proliferation, metastases and tumor sensitivity

Abstract Background: It has been proposed that therapy-induced overexpression of ATP-binding cassette (ABC) drug efflux pumps promotes resistance in many drug-treated cancers. While early studies focused on overexpression and activity of the P-glycoprotein (Pgp) pump, a structurally distinct group of ABC efflux pumps, known as the Multidrug Resistance Proteins (MRPs;ABCCs), have been gaining increasing attention as alternative sources of resistance. There are 9 ABCC family members (ABCC1-6, 10–12), and we have previously shown that ABCC10 is unique because it confers resistance to a wide variety of anticancer agents including taxanes, vinca alkaloids and nucleoside-based analogs (Hopper-Borge et al, 2004, Hopper-Borge et al, 2009). Importantly we have shown that Abcc10 null mice are sensitized to taxanes (Hopper-Borge et al, Cancer Research, 2011). This is the first transporter whose loss results in in vivo tissue sensitivity to taxanes. Methods & Results: We recently bred a PyVmT mammary tumor model to our Abcc10 gene disrupted mice to investigate Abcc10's in vivo activities regarding breast cancer resistance mechanisms. We have observed that Abcc10+/+ (WT) status limits the rate of tumorigenesis (p = 0.020) and that WT tumors are more metastatic compared to Abcc10−/− (KO), (p = 0.0489). To better understand the potential resistance mechanisms in this model we determined the expression levels of relevant transporters with qRT-PCR and found no significant differences between WT and KO. The PI3K-AKT-mTOR, a major signaling pathway involved in breast cancer progression has been connected to ABCCs (Tazzari, et al, 2007), and we observed upregulation of AKT in KO versus WT mammary tumor cell lines via western analyses. Therefore, we decided to ask if Abcc10 status affects proliferation, attachment, and/or migration (CyQuant, Invitrogen and xCELLigence, Roche). We showed that WT cell lines grow 3 times slower compared to KO. For migration kinetic we observed that KO cell lines were more active compared to WT. To determine the impact of Abcc10 loss in vivo we injected SCID mice with 3 WT or KO mammary tumor derived cell lines and treated tumors with docetaxel or vehicle. Tumors derived from WT and KO mice were both responsive to docetaxel (WT, p = 0.003 and KO, p = 0.001). However, over 21 days, the WT tumors were reduced by 50% compared to KO reduction of 86%. Kaplan-Meier survival analysis of treated/untreated SCID mice reveals that Abcc10 status is related to a poorer prognosis (KO p < 0.0001, WT p = 0.0648). Conclusion: Here we show that Abcc10 status limits tumorigenesis (proliferation, attachment, migration) of MMTV-PyVmT mammary tumor model and Abcc10+/+ tumors are more metastatic compared to KO tumors. Thus Abcc10 may be involved in breast cancer development and progression. mRNA expression analysis of ABC transporters in mammary tumors derived cell lines reveals that other transporters do not compensate for Abcc10 loss. In vivo experiments show that treatment with docetaxel is more effective for KO tumors compared to WT tumors, suggesting Abcc10 is a breast cancer resistance factor. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-04.

Overexpression of the Protein Tyrosine Phosphatase PRL-2 Correlates with Breast Tumor Formation and Progression

The PRL-1, PRL-2, and PRL-3 phosphatases are prenylated protein tyrosine phosphatases with oncogenic activity that are proposed to drive tumor metastasis. We found that PRL-2 mRNA is elevated in primary breast tumors relative to matched normal tissue, and also dramatically elevated in metastatic lymph nodes compared with primary tumors. PRL-2 knockdown in metastatic MDA-MB-231 breast cancer cells decreased anchorage-independent growth and cell migration, suggesting that the malignant phenotype of these cells is mediated at least in part through PRL-2 signaling. In different mouse mammary tumor-derived cell lines overexpressing PRL-2, we confirmed its role in anchorage-independent growth and cell migration. Furthermore, injection of PRL-2-overexpressing cells into the mouse mammary fat pad promoted extracellular signal-regulated kinase 1/2 activation and tumor formation. MMTV-PRL-2 transgenic mice engineered to overexpress the enzyme in mammary tissue did not exhibit spontaneous tumorigenesis, but they exhibited an accelerated development of mammary tumors initiated by introduction of an MMTV-ErbB2 transgene. Together, our results argue that PRL-2 plays a role in breast cancer progression.

Abstract 4144: The treatment with a derivative of Vitamin A (ATRA) modulates the expression and subcellular localization of Protein Kinase C (PKC) delta and induces growth inhibition in normal and tumor derived mammary cells

Abstract Retinoids may exert some of their effects on cell differentiation and malignant phenotype reversion through the interaction with different PKC isoforms. In the present work we addressed the following issues: A)- the role of ATRA on the in vitro growth potential of LM3, a murine mammary tumor-derived cell line; NMuMG, a normal murine mammary cell line and MDA-MB231, a human breast cancer-derived cell line. B)- the effect of retinoid treatment on the expression and subcellular distribution of PKCδ and C)- the importance of PKCδ in the activity of retinoid receptors. ATRA treatment (1μM, 72h) induced a significant growth inhibition in the analyzed murine cell lines. This phenomenon was associated with the reduction of pERK1 levels (80±9% and 40±5% in LM3 and NMuMG cells respectively) and the increase of the cell cycle inhibitor p27 in the nuclear fraction without altering the expression of Cyclin D1 in both murine cell lines. None of these modulations were observed in the ATRA unresponsive MDA-MB241 cell line. By Western blot we could determine that 72h treatment with ATRA induced an increase of PKCδ protein levels both in the nuclear fraction of LM3 cells (4-fold) and in the cytoplasm (2- and 3-fold for LM3 and NMuMG cells respectively). On the contrary, in MDA-MB241 cells PKCδ expression was reduced after ATRA exposure. Interestingly, pharmacological silencing of PKCδ in LM3 cells prevented the activation of retinoid receptors by ATRA, as evidenced by a reporter gene assay (RARE-Luciferase). Our results suggest that, in ATRA-responsive cells, the treatment with this vitamin A derivate inhibits in vitro growth, by reducing mitogenic signaling, by increasing the expression of a cell-cycle inhibitory protein as well as by increasing PKCδ an isoform usually associated with growth inhibition and apoptosis induction. The subcellular localization was also altered by this treatment in LM3 cells. Moreover, the effect of ATRA exerted through the activation of its specific nuclear receptors would depend on the nuclear translocation of this PKC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4144.

PKCθ promotes c-Rel–driven mammary tumorigenesis in mice and humans by repressing estrogen receptor α synthesis

The vast majority of primary human breast cancer tissues display aberrant nuclear NF-kappaB c-Rel expression. A causal role for c-Rel in mammary tumorigenesis has been demonstrated using a c-Rel transgenic mouse model; however, tumors developed with a long latency, suggesting a second event is needed to trigger tumorigenesis. Here we show that c-Rel activity in the mammary gland is repressed by estrogen receptor alpha (ERalpha) signaling, and we identify an epigenetic mechanism in breast cancer mediated by activation of what we believe is a novel PKCtheta-Akt pathway that leads to downregulation of ERalpha synthesis and derepression of c-Rel. ERalpha levels were lower in c-Rel-induced mammary tumors compared with normal mammary gland tissue. PKCtheta induced c-Rel activity and target gene expression and promoted growth of c-Rel- and c-RelxCK2alpha-driven mouse mammary tumor-derived cell lines. RNA expression levels of PKCtheta and c-Rel target genes were inversely correlated with ERalpha levels in human breast cancer specimens. PKCtheta activated Akt, thereby inactivating forkhead box O protein 3a (FOXO3a) and leading to decreased synthesis of its target genes, ERalpha and p27(Kip1). Thus we have shown that activation of PKCtheta inhibits the FOXO3a/ERalpha/p27(Kip1) axis that normally maintains an epithelial cell phenotype and induces c-Rel target genes, thereby promoting proliferation, survival, and more invasive breast cancer.

Paradoxical antiproliferative effect by a murine mammary tumor-derived epithelial cell line

BackgroundDespite significant advancement in breast cancer therapy, there is a great need for a better understanding of the mechanisms involved in breast carcinogenesis and progression, as well as of the role of epigenetic contributions from stromal cells in mammary tumorigenesis. In this study, we isolated and characterized murine mammary tumor-derived epithelial and myofibroblast cell lines, and investigated the in vitro and in vivo effect of cellular soluble factors produced by the epithelial cell line on tumor cells.MethodsMorphology, immunophenotype, cytogenetics, invasiveness, and tumorigenicity of epithelial (LM-234ep) and myofibroblast (LM-234mf) cell lines isolated from two murine mammary adenocarcinomas with common ancestor were studied. The in vitro effects of LM-234ep conditioned medium on proliferation, cell cycle distribution, and expression of cell cycle proteins, were investigated in LM-234mf cells, mouse melanoma cells (B16-F10), and human cervical adenocarcinoma cells (HeLa). The in vivo anti-tumor activity of LM-234ep conditioned media was evaluated in subcutaneous tumors formed in nude mice by B16-F10 and HeLa cells.ResultsLM-234ep cells were found to be cytokeratin positive and hipertriploid, whereas LM-234mf cells were α-smooth muscle actin positive and hypohexaploid. Chromosome aberrations were found in both cases. Only LM-234mf revealed to be invasive in vitro and to secrete active MMP-2, though neither of the cell types were able to produce progressing tumors. LM-234ep-derived factors were able to inhibit the in vitro growth of LM-234mf, B16-F10, and HeLa cells, inducing cell cycle arrest in G0/G1 phase. The administration of LM-234ep conditioned medium inhibited the growth of B16-F10 and HeLa tumors in nude mice.ConclusionOur data suggest the existence of epithelial cell variants with tumor suppressive properties within mammary tumors. To our knowledge, this is the first report showing antiproliferative and antineoplastic activities induced by tumor-derived epithelial cells.

EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF MOUSE CYP2D22

Metabolism of the prototype human CYP2D6 substrates debrisoquine and bufuralol proceeds at a much slower rate in mice; therefore, the mouse has been proposed as an animal model for the human CYP2D6 genetic deficiency. To interpret the molecular mechanism of this deficiency, a cDNA belonging to the CYP2D gene subfamily (Cyp2d22) has been cloned and sequenced from a mouse mammary tumor-derived cell line. In the current study, Cyp2d22 enzyme was overexpressed and purified from insect cells using a baculovirus-mediated system. The activity of this purified enzyme was directly compared with purified human CYP2D6 toward codeine, dextromethorphan, and methadone as substrates. Purified Cyp2d22 was found to catalyze the O-demethylation of dextromethorphan with significantly higher K(m) values (250 microM) than that (4.2 microM) exhibited by purified human CYP2D6. The K(m) for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 microM, much lower than that observed with human CYP2D6 and near the K(m) for dextromethorphan N-demethylation catalyzed by CYP3A4. CYP2D6 catalyzed codeine O-demethylation, whereas Cyp2d22 and CYP3A4 mediated codeine N-demethylation. Furthermore, methadone, a known CYP3A4 substrate and CYP2D6 inhibitor, was N-demethylated by Cyp2d22 with a K(m) of 517 microM and V(max) of 4.9 pmol/pmol/min. Quinidine and ketoconazole, potent inhibitors to CYP2D6 and CYP3A4, respectively, did not show strong inhibition toward Cyp2d22-mediated dextromethorphan O- or N-demethylation. These results suggest that mouse Cyp2d22 has its own substrate specificity beyond CYP2D6-like-deficient activity.

Inefficient proteasomal‐degradation pathway stabilizes AP‐2α and activates HER‐2/neu gene in breast cancer

HER-2/neu proto-oncogene is overexpressed in about one fourth of human breast cancers. AP-2 transcription factors bind to the HER-2/neu gene promoter and activate its expression. In a striking concurrence, anomalous abundance of AP-2alpha protein or its homolog AP-2gamma is also detected with HER-2/neu protein in mammary tumor-derived cell lines. This suggests that the deregulation of AP-2 is the preceding pathogenic event and probably the pivotal one in this type of mammary carcinogenesis. We examined the process of AP-2alpha gene expression in mammary carcinoma cell lines to identify where the aberration had occurred. We found no amplification of the AP-2alpha gene. Its promoter was marginally upregulated; however, it did not significantly increase the mRNA levels. When the AP-2alpha protein was examined, a remarkable stability was seen in breast cancer cell lines MDA-MB-453 and SK-BR-3, with a half-life of over 30 hr. This is sharply higher than the approximate 1 hr observed in mammary epithelial cell line MCF-10A and murine cell line NIH 3T3. Treatment of MCF-10A and NIH 3T3 cells with the proteasome inhibitor MG-132 showed that AP-2alpha was ubiquitinated and its level significantly increased. Moreover, this increase was accompanied by elevated levels HER-2/neu protein. In contrast, weaker ubiquitination of AP-2alpha was seen in MDA-MB-453 and SK-BR-3 cancer cells, and MG-132 treatment did not raise the AP-2alpha level any further. These results uncover that unusual stability is the main mechanism that raises the levels of AP-2 proteins, and in addition, provide the first clue that defective ubiquitin-dependent proteasomal-degradation pathway is possibly the prime cause that affects the HER-2/neu gene and culminates in breast cancer.

Cyclin D1 is necessary but not sufficient for anchorage-independent growth of rat mammary tumor cells and is associated with resistance of the Copenhagen rat to mammary carcinogenesis.

To identify genes associated with the resistance of Copenhagen (Cop) rats to mammary carcinogenesis, we infused a retrovirus harboring v-Ha-ras directly into the main mammary ducts of resistant F1 rats from a cross between Cop and susceptible Wistar Furth (WF) rats. Adenocarcinomas formed in approximately 50% of infused glands. Cell lines derived from these tumors were clonal, but did not share a common viral integration site, suggesting that a high level of v-Ha-ras expression was able to overcome resistance in the F1 rats. Some of the cell lines were able to grow in soft agar, but a significant number did not display anchorage-independent growth. These growth characteristics were independent of v-Ha-ras expression levels. The ability to grow in soft agar was associated with the size of tumors induced by injecting the cells into nude mice, and showed a striking positive association with the expression of cyclin D1. Furthermore, while resistance to anchorage-independent growth was fully overcome by transfection of cyclin D1 in some clones, in the others the effect was partial. A similar pattern of cyclin D1 upregulation and growth in soft agar was also observed when the cells were transfected with an active form of beta-catenin. Hybrid cells from the somatic fusion of an anchorage-dependent to an anchorage-independent clone did not grow in soft agar. These results suggest that while a high expression level of cyclin D1 is necessary for anchorage-independent growth in all clones, it is not sufficient for full growth capacity in soft agar, raising the possibility that the loss of a tumor suppressor gene in the cell lines is required to fully confer anchorage-independent growth. Our anchorage-dependent and -independent rat mammary tumor-derived cell lines may recapitulate the resistance and susceptibility of Cop and WF rats, respectively, to mammary carcinogenesis that could facilitate the identification of breast cancer susceptibility genes.

Lipoprotein assembly capacity of the mammary tumor-derived cell line C127 is due to the expression of functional microsomal triglyceride transfer protein

C127, a murine mammary tumor-derived cell line, is capable of lipidating and secreting apolipoprotein B-41 (apoB-41) in the apparent absence of microsomal triglyceride transfer protein (MTP). Using a semiquantitative reverse transcriptase-coupled polymerase chain reaction, mouse MTP mRNA was detected in C127 cells at ~10–20% of the relative abundance of human MTP in HepG2 cells. Radiolabeling of C127 cells with [35S]methionine and [35S]-cysteine followed by immunoprecipitation with anti-MTP antibodies identified a band with an electrophoretic mobility identical to that of authentic mouse MTP. Cotransfection of apoB-41 and the MTP 97-kDa subunit in C127 cells enhanced apoB secretion by ~5-fold relative to apoB-41 transfection alone, suggesting that MTP is limiting in these cells. To establish that MTP expression is responsible for apoB-containing lipoprotein assembly in C127 cells, the effects of the MTP inhibitor BMS-200150 were examined. Secretion of apoB-41 by C127 cells was inhibited to the same extent observed in COS-1 cells cotransfected with apoB-41 and MTP. These results suggest that low MTP expression, and not the expression or overexpression of another known or novel factor(s), is responsible for apoB assembly and secretion in C127 cells and further supports the essential nature of MTP in the biogenesis of apoB-containing lipoproteins.—Sellers, J. A., and G. S. Shelness. Lipoprotein assembly capacity of the mammary tumor-derived cell line C127 is due to the expression of functional microsomal triglyceride transfer protein.

Characterization of isotype-specific regions of five classes of canine beta-tubulin and their expression in several tissues and cell culture.

The structure of isotype-specific regions of classes 1, II, III, IVa and IVb of canine beta-tubulin was characterized by 3'-RACE and the expression of these isotypes in canine tissues was examined by ribonuclease protection assay (RPA). Furthermore, a malignant mammary tumor-derived osteosarcoma-like cell line was established and the altered expression of beta-tubulin isotypes in taxol-resistant sublines was analyzed. The deduced amino acid sequences in isotype-specific regions corresponding to classes I, II and IVb were identical to those of humans and mice, but those in classes III and IVa showed slight differences among species. RPA revealed that classes I and IVb were widely distributed, but classes II, III and IVa were restricted to the brain. Because RPA could clearly distinguish the expression of class IVa from that of class IVb, it was thought to be more useful than northern blot for analysis of beta-tubulin isotype expression. In vitro, taxol-resistant sublines displayed a significant increase in class IVa as compared with taxol-sensitive cells, suggesting that altered expression of class IVa was associated with taxol resistance in these cell lines.

Characterization of Cyp2d22, a Novel Cytochrome P450 Expressed in Mouse Mammary Cells

Endogenous steroids and numerous environmental agents have potent effects on mammary development and carcinogenesis. Locally produced cytochrome P450 enzymes that modify such molecules are therefore likely to be important regulators of these processes. Here we describe the characterization of a novel mouse gene, termed Cyp2d22, that is highly expressed in the mammary tumor derived cell line RIII/Pr1. Cyp2d22 is expressed at intermediate levels in the weakly tumorigenic cell line RIII/MG, whereas expression is low or absent in all normal mouse mammary epithelial cell lines tested and three C3H mammary tumor derived cell lines. Immunoblot analysis of mouse tissues with highly specific antisera indicates that 2D22 protein levels are most abundant in liver, while intermediate levels of expression are seen in adrenal, ovary, and mammary gland. Immunohistochemical staining of liver sections with these antisera demonstrates that 2D22 is most abundant in the first layer or two of parenchymal cells surrounding the central vein, with virtually no expression detected in periportal cells. Interestingly, sequence similarity and functional data suggest that Cyp2d22 may be the mouse ortholog of human CYP2D6. These observations support the hypothesis that 2D22 mediates a distinct, biologically significant activity in relation to other mouse 2D family members.

Mapping of a mouse mammary tumor virus integration site by retroviral LTR—arbitrary polymerase chain reaction

The de novo integration of retroviral genomes within the mammalian genome is believed to contribute to the tumorigenic process. Integration may result in the disruption or inappropriate transcription of key regulatory genes. We describe the application of an arbitrarily primed PCR method for the mapping and cloning of genomic integration sites of the mouse mammary tumor virus (MMTV). We have amplified DNA sequences between a selected retroviral MMTV-LTR and random sites in the 3′ flanking DNA. Using this technique we were able to visualize several proviral integration sites present in a MMTV-induced mammary tumor derived cell line that were absent from the germ line. Cloning and sequencing of the PCR product corresponding to one site established its identification as an unique 3′ flanking sequence.

Expression of a 32 kDa protein in rat mammary tumors induced by anti-benzo[c]phenanthrene-3,4-diol-1,2-epoxide.

Racemic anti-benzo[c]phenanthrene-3,4-diol-1,2-epoxide (BcPDE) is a powerful rat mammary carcinogen and a metabolite of benzo[c]phenanthrene, a polynuclear aromatic hydrocarbon found in the environment. In elucidating potential molecular mechanisms that may play a role in the development of BcPDE-induced rat mammary tumors, we have identified a 32 kDa protein in 16 of 26 tumors analyzed but in only 1 of the 15 normal mammary tissues that were examined. The 32 kDa protein was identified with antibodies to Ets, which also recognized the 55 kDa Ets-1 protein that was expressed at similar levels in normal mammary tissues. The expression of the 32 kDa protein was also observed in mammary tumor-derived cell lines of both rat and human origin and in human melanoma, but not in normal human keratinocytes or rat fibroblast cell lines. Further characterization via 2D gels revealed that the protein exhibits a PI of 5.5. Southwestern analysis using Ets-1 target sequence revealed binding of the 55 kDa Ets-1 but not of the newly identified 32 kDa protein. Overall, the preferential expression of the 32 kDa protein in mammary tumor tissues may serve as a biomarker to follow the development of this tumor type.

Expression of the oncogene c-erbB-2 in canine mammary cancers and tumor-derived cell lines

Abstract Objective To determine, for canine mammary tumors, whether malignancy, with or without local invasion or regional metastasis, was associated with overexpression of the oncogene c-erbB-2. Design c-erbB-2 expression was measured in canine mammary tumor-derived cell lines and in mammary tumor tissues from clinical cases. Clinical samples were examined histologically to determine whether they were benign or malignant and, if malignant, whether they had evidence of local invasion or regional metastasis. Canine fibroblast cultures and normal canine mammary epithelial tissues were used as reference standards for cell lines and mammary tumors, respectively. Sample Populations 28 canine mammary tumor tissue samples obtained surgically from clinical cases and samples from 7 canine mammary tumor cell lines derived from primary canine mammary tumors. Procedure c-erbB-2 mRNA levels were determined by means of hybridization of total polysomal RNA with a 32P-labeled human c-erbB-2 probe on dot blots, and results were quantified by means of scanning densitometry. Overexpression of c-erbB-2 was defined as an autoradiographic density ≥ 2 times the density of reference samples on the same blot. Results Overexpression of c-erbB-2 was detected in 17 of 23 malignant tumors, 0 of 5 benign tumors, and 2 of 7 mammary tumor cell lines. c-erbB-2 overexpression was correlated with a histopathologic diagnosis of malignancy (P= 0.005) but not with the presence of local invasion or regional metastatic disease (P = 0.621). Conclusions Results suggest that overexpression of c-erbB-2 occurs prior to the development of metastatic disease in canine mammary tumors and plays a role in the development of malignancy. (Am J Vet Res 1996; 57:693–696)

Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells.

Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991 a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrine growth factor to support the EGF-independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR. The EGF-independent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-EGF receptor antibody to the culture medium, implicating AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-alpha) mRNA revealed coordinate expression of AR and TGF-alpha in these cells. These data suggest that both AR and TGF-alpha mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs.