Dominant negative retinoic acid receptor initiates tumor formation in mice

Background  

Retinoic acid suppresses cell growth and promotes cell differentiation, and pharmacological retinoic acid receptor (RAR) activation is anti-tumorigenic. This begs the question of whether chronic physiological RAR activation by endogenous retinoids is likewise anti-tumorigenic.     

Dominant negative EGFR-CD533 and inhibition of MAPK modify JNK1 activation and enhance radiation toxicity of human mammary carcinoma cells.

Exposure of MDA-MB-231 human mammary carcinoma cells to an ionizing radiation dose of 2 Gy results in immediate activation and Tyr phosphorylation of the epidermal growth factor receptor (EGFR). Doxycycline induced expression of a dominant negative EGFR-CD533 mutant, lacking the COOH-terminal 533 amino acids, in MDA-TR15-EGFR-CD533 cells was used to characterize intracellular signaling responses following irradiation. Within 10 min, radiation exposure caused an immediate, transient activation of mitogen activated protein kinase (MAPK) which was completely blocked by expression of EGFR-CD533. The same radiation treatment also induced an immediate activation of the c-Jun-NH2-terminal kinase 1 (JNK1) pathway that was followed by an extended rise in kinase activity after 30 min. Expression of EGFR-CD533 did not block the immediate JNK1 response but completely inhibited the later activation. Treatment of MDA-TR15-EGFR-CD533 cells with the MEK1/2 inhibitor, PD98059, resulted in approximately 70% inhibition of radiation-induced MAPK activity, and potentiated the radiation-induced increase of immediate JNK1 activation twofold. Inhibition of Ras farnesylation with a concomitant inhibition of Ras function completely blocked radiation-induced MAPK and JNK1 activation. Modulation of EGFR and MAPK functions also altered overall cellular responses of growth and apoptosis. Induction of EGFR-CD533 or treatment with PD98059 caused a 3-5-fold increase in radiation toxicity in a novel repeated radiation exposure growth assay by interfering with cell proliferation and potentiating apoptosis. In summary, this data demonstrates that both MAPK and JNK1 activation in response to radiation occur through EGFR-dependent and -independent mechanisms, and are mediated by signaling through Ras. Furthermore, we have demonstrated that radiation-induced activation of EGFR results in downstream activation of MAPK which may affect the radiosensitivity of carcinoma cells.     

Ethanol inhibits benzo[a]pyrene-DNA adduct removal and increases 8-oxo-deoxyguanosine formation in human mammary epithelial cells

The effect of ethanol and acetaldehyde treatment on the removal of benzo[a]pyrene diol-epoxide (BPDE)-DNA adducts in the immortalized, human mammary epithelial cell line MCF-10F was examined. Treatment of cells with 15 mM and 25 mM ethanol resulted in significantly more BPDE-DNA adducts/unit DNA remaining at multiple time points, compared to controls. The half-life of BPDE-DNA adducts in cells exposed to both 15 and 25 mM ethanol were 11.9 and 12.3 h, respectively, compared to a half-life of 9.8 h for the control cells. In contrast, for cells exposed to acetaldehyde at doses of 2.5 and 5.0 microM no significant trend in BPDE-DNA adduct persistence occurred, compared to controls. The inhibition of adduct removal for cells treated with ethanol was not associated with any changes in cell viability due to ethanol exposure. However, BP-treated cells exposed to 25 mM ethanol exhibited a significant 2-fold increase in 8-oxo-deoxyguanosine (8-oxo-deG) adducts compared to BP-treated cells alone. No significant increase in 8-oxo-deG was observed for cells treated with BP and exposed to 5.0 microM acetaldehyde. Thus, ethanol exposure of human mammary epithelial cells is associated with a decreased capacity to remove BPDE-DNA adducts. This inhibitory effect of ethanol on adduct removal in part may be related to ethanol-associated oxidative stress.     

PARP inhibitor reduces proliferation and increases apoptosis in breast cancer cells

Objective： Apoptosis is a reliable marker of chemotherapeutic efficacy. Olaparib and paclitaxel inhibit proliferation and induce apoptosis in a variety of cancers. We investigated the effects of paclitaxel combined with olaparib on apoptosis in breast cancer Bcap37 cells. Methods： Proliferation and apoptosis were detected by MTT assay and PI staining. Degradation of procaspase-3 and poly（ADP-ribose） polymerase （PARP） was analyzed by Western blotting. Results： Compared with paclitaxel alone, paclitaxel combined with 100 mg olaparib significantly reduced survival in Bcap37 cells at all tested treatment durations （P〈0.05）; inhibition increased with increasing olaparib dose and treatment time （P〈0.01）. Combined treatment yielded significantly higher rates of apoptosis （P〈0.05）, which also increased with time （P〈0.01）. Fluorescence micrographs showed that early and late apoptotic cells increased with treatment time. Pro-caspase-3 and PARP degradation was induced by paclitaxel and enhanced by olaparib in a dose-dependent manner. Thus, combined treatment was substantially more effective than treatment with paclitaxel alone. Conclusions： Our findings suggest that paclitaxel and olaparib inhibit breast cancer Bcap37 cell proliferation and induce apoptosis. Combined treatment further reduced cell growth and enhanced apoptosis, suggesting that this combination therapy may be a promising treaunent for breast cancer.     

Dominant negative MEKK1 inhibits survival of pancreatic cancer cells

Human pancreatic cancers harbor mutations in the K-ras gene, and these mutations convert the gene oncogenic and constitutively active forms. However, in pancreatic cancer cells little is known about the activation of the downstream pathways of Ras, MEK-ERK and MEKK1-JNK, and their roles in cell survival and proliferation. An analysis of nine pancreatic cancer tissues revealed JNK activation in all tumor samples and ERK activation in three tumor samples. Colony formation assays by transfection of dominant negative mutants of Ras, ERK or MEKK1 into pancreatic cancer cell lines (BxPC-3, PANC-1, MIAPaCa-2 and AsPC-1) and an amnion-derived cell line (FL) revealed that DN-MEKK strongly inhibits the survival of colonies in pancreatic cancer cells, but not in FL cells. In vitro kinase assays and luciferase assays using the Gal4c-Jun system revealed that in pancreatic cancer cells DN-MEKK fails to inhibit JNK activation. In PANC-1 cells, c-Jun was found to be a major component of protein component binding to AP-1 site and CRE, but not in FL cells. The inhibitory effect of DN-MEKK in PANC-1 cells was thought to be the result of the inhibition of c-Jun DNA-binding. The difference of suppression in pancreatic cancer cells and non-pancreatic cancer cells suggested that the MEKK1 pathway mainly contributes to cell survival in pancreatic cancer cells and may provide an advantage for the gene therapy of pancreatic cancers using DN-MEKK expression vectors.     

小管形成实验在肿瘤微血管形成中的应用研究

肿瘤的增殖与侵袭有赖于肿瘤新生微血管的形成,破坏或者抑制此过程就能切断肿瘤生长的“营养与能源”,因此肿瘤血管生成抑制因子、抗血管生成的药物及研究方法已成为人们研究的热点。本文就小管形成实验在肿瘤微血管生成的应用及影响小管实验管腔形成的各项因素做一综述。     

Defibrotide reduces procoagulant activity and increases fibrinolytic properties of endothelial cells.

Defibrotide (DF), a polydeoxyribonucleotide with antithrombotic properties, has recently proven effective in patients with severe hepatic veno-occlusive disease (VOD), a life-threatening complication of high-dose chemo/radiotherapy regimens for stem cell transplantation. To understand the mechanism of its beneficial effect, we studied the impact of DF on the expression of tissue factor (TF) and fibrinolytic proteins (PAI-1 and t-PA) on endothelial cells. The in vitro response to DF of two types of human endothelial cells (ECs) of different origins, that is from macrovascular (HUVEC) and microvascular (HMEC-1 cell line) beds, was evaluated in the presence or absence of a proinflammatory stimulus (ie bacterial endotoxin, LPS). The results show that DF was able to significantly reduce the LPS-induced TF expression by HMEC-1, and less prominently by HUVEC. In addition, DF importantly influenced the fibrinolytic properties of both HMEC-1 and HUVEC. Specifically, it dose-dependently counteracted the LPS-induced increase in PAI-1 levels and decrease in t-PA activity expression. It also significantly incremented t-PA antigen in resting EC. Decreasing the procoagulant activity and increasing the fibrinolytic potential of EC favors an anticoagulant phenotype of the endothelium, which may protect from fibrin deposition and vascular occlusion.     

Dominant negative ATM mutations in breast cancer families

BACKGROUND: The ATM gene encoding a putative protein kinase is mutated in ataxia-telangiectasia (A-T), an autosomal recessive disorder with a predisposition for cancer. Studies of A-T families suggest that female heterozygotes have an increased risk of breast cancer compared with noncarriers. However, neither linkage analyses nor mutation studies have provided supporting evidence for a role of ATM in breast cancer predisposition. Nevertheless, two recurrent ATM mutations, T7271G and IVS10-6T-->G, reportedly increase the risk of breast cancer. We examined these two ATM mutations in a population-based, case-control series of breast cancer families and multiple-case breast cancer families. METHODS: Five hundred twenty-five or 262 case patients with breast cancer and 381 or 68 control subjects, respectively, were genotyped for the T7271G and IVS10-6T-->G ATM mutations, as were index patients from 76 non-BRCA1/2 multiple-case breast cancer families. Linkage and penetrance were analyzed. ATM protein expression and kinase activity were analyzed in lymphoblastoid cell lines from mutation carriers. All statistical tests were two-sided. RESULTS: In case and control subjects unselected for family history of breast cancer, one case patient had the T7271G mutation, and none had the IVS10-6T-->G mutation. In three multiple-case families, one of these two mutations segregated with breast cancer. The estimated average penetrance of the mutations was 60% (95% confidence interval [CI] = 32% to 90%) to age 70 years, equivalent to a 15.7-fold (95% CI = 6.4-fold to 38.0-fold) increased relative risk compared with that of the general population. Expression and activity analyses of ATM in heterozygous cell lines indicated that both mutations are dominant negative. CONCLUSION: At least two ATM mutations are associated with a sufficiently high risk of breast cancer to be found in multiple-case breast cancer families. Full mutation analysis of the ATM gene in such families could help clarify the role of ATM in breast cancer susceptibility.     

Fibrinogen E-fragment inhibits the migration and tubule formation of human dermal microvascular endothelial cells in vitro

Angiogenesis, the development of new blood vessels from an existing vascular bed, is essential for the growth and spread of malignant tumors. Several endogenous angiogenesis inhibitors have been discovered and shown to suppress endothelial cell function in vitro and tumor growth in vivo. Several of these are proteolytic fragments of larger, endogenous proteins. Here we show that a Mr 50,000 polypeptide derived from the plasmin cleavage of fibrinogen, fibrinogen E-fragment, inhibits endothelial cell migration and tubule formation induced by both proangiogenic growth factors, vascular endothelial growth factor and basic fibroblast growth factor, in vitro.     

Aurora-A, a negative prognostic marker, increases migration and decreases radiosensitivity in cancer cells

Centrosomal Aurora-A (Aur-A) kinase ensures proper spindle assembly and accurate chromosome segregation in mitosis. Overexpression of Aur-A leads to centrosome amplification, aberrant spindle, and consequent genetic instability. In the present study, Aur-A was found to be overexpressed in laryngeal squamous cell carcinoma (LSCC). Moreover, Aur-A expression was adversely correlated with median survival, and further identified as a potential independent factor for disease prognosis. Suppression of Aurora kinase activity chemically or genetically led to LSCC Hep2 cell cycle arrest and apoptotic cell death. Importantly, we found that Aur-A increases cell migration and this novel function was correlated with Akt1 activation. The enhanced cell migration induced by Aur-A overexpression could be abrogated by either small-molecule Akt1 inhibitor or short interfering RNA. VX-680, a selective Aurora kinase inhibitor, decreased Akt1 phosphorylation at Ser(473) and inhibited cell migration, but failed to do so in constitutive active Akt1 (myr-Akt1)-overexpressed cells. Moreover, our data suggested that overexpression of Aur-A kinase might also contribute to radioresistance of LSCC. Inhibiting Aur-A by VX-680 induced expression of p53 and potently sensitized cells to radiotherapy, leading to significant cell death. Ectopic overexpression of Aur-A, however, reduced p53 level and rendered cells more resistant to irradiation. Taken together, we showed that Aur-A kinase, a negative prognostic marker, promotes migration and reduces radiosensitivity in laryngeal cancer cells.     

Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells

Expression of the chemokine receptor CXCR4 has been linked with increased metastasis and decreased clinical prognosis in breast cancer. The current paradigm dictates that CXCR4 fosters carcinoma cell metastasis along a chemotactic gradient to organs expressing the ligand CXCL12. The present study asked if alterations in autocrine CXCR4 signaling via dysregulation of CXCL12 in mammary carcinoma cells modulated their metastatic potential. While CXCR4 was consistently detected, expression of CXCL12 characteristic of human mammary epithelium was silenced by promoter hypermethylation in breast cancer cell lines and primary mammary tumors. Stable re-expression of functional CXCL12 in ligand null cells increased orthotopic primary tumor growth in the mammary fat-pad model of tumorigenesis. Those data parallel increased carcinoma cell proliferation measured in vitro with little-to-no-impact on apoptosis. Moreover, re-expression of autocrine CXCL12 markedly reduced metastatic lung invasion assessed using in vivo bioluminescence imaging following tail vein injection. Consistent with those data, decreased metastasis reflected diminished intracellular calcium signaling and chemotactic migration in response to exogenous CXCL12 independent of changes in CXCR4 expression. Together these data suggest that an elevated migratory signaling response to ectopic CXCL12 contributes to the metastatic potential of CXCR4-expressing mammary carcinoma cells, subsequent to epigenetic silencing of autocrine CXCL12.     

Basal lamina formation by normal and transformed mouse mammary epithelial cells duplicated in vitro

Cells from low-passage (LP) cultures of a mouse mammary epithelial line (NMuMG cells) form a basal lamina when they are cultured on a type I collagen gel substratum. A high-passage (HP) strain of this line maintained the morphologic, serologic, and karyologic properties of the LP cells. For the determination of whether transformation of the NMuMG cells might lead to defects in the basal lamina, cells from LP cultures were compared in vivo and in vitro with cells of HP cultures for tumorigenicity, growth characteristics, and ability to form a lamina. The LP NMuMG cells had a typical epithelial morphology and showed no cytologic evidence of cancer. They formed an ultrastructurally normal continuous basal lamina in vivo when they were injected into athymic nude mice. In contrast, the HP cells were pleomorphic and highly invasive when injected into nude mice where they showed frequent and large basal lamina defects. These cells also accumulated only traces of lamina-like materials when cultured on a collagen gel, indicating that neoplastic transformation had markedly reduced the ability of NMuMG cells to form a basal lamina both in vivo and in vitro. Because the collagen gel culture system duplicated the in vivo situation with regard to basal lamina integrity, the basis for this lack of in vitro basal lamina formation may be physiologically relevant for the mechanism of malignant invasion.     

High birth weight increases mammary tumorigenesis in rats

Epidemiological studies have investigated whether a high birth weight is associated with increased breast cancer risk, but the results remain inconclusive. This study was designed to determine whether high birth weight increases later susceptibility to carcinogen-induced mammary tumorigenesis in an animal model and to determine mechanisms mediating this association. Pregnant female Sprague Dawley rats were fed either a control or a high-fat diet during the extent of gestation. Maternal exposure to the high-fat diet increased pregnancy leptin levels and offspring's birth weight, but had no effect on pregnancy estradiol or insulin-like growth factor 1 levels. Changes in the offspring's mammary gland morphology and protein expression were assessed. The mammary epithelial tree of the high-birth-weight offspring was denser, contained more terminal end buds and exhibited higher number of proliferating cells. Further, their mammary glands expressed lower levels of ER-alpha, but higher levels of activated MAPK. No alterations in apoptosis were noted. High-birth-weight rats developed 7,12-dimethylbenz[a]anthracene-induced mammary tumors significantly earlier, and tumors grew larger than in the controls. The tumors in this group expressed higher levels of leptin receptor and activated Akt, and contained fewer apoptotic cells than those in the controls. Our results indicate that high birth weight is related to shortened latency to develop mammary tumors--perhaps reflecting an increase in activated MAPK levels and increased tumor growth--perhaps caused by a lower apoptotic response due to higher leptin receptor and activated Akt levels in the tumors.     

Caloric restriction reduces growth of mammary tumors and metastases

We investigated the effects of caloric restriction (CR) on growth of tumors and metastases in the 4T1 mammary tumor model and found that CR, compared with normal diet, reduced the growth of mammary tumors and metastases and the total number of metastases that originated both spontaneously from the primary tumor and also experimentally from i.v. injection of the tumor cells. CR also decreased proliferation and angiogenesis and increased apoptosis in tumors. CR reduced levels of insulin, leptin, insulin-like growth factor 1, insulin-like growth factor binding protein 3 and increased adiponectin in tumors. We also demonstrated that tumors from CR mice possessed lower levels of transforming growth factor-β, lower intratumor deposition of collagen IV and reduced invasiveness due to a decrease in tumor secretion of active matrix metalloproteinase 9. Our results suggest that CR-induced metabolic and signaling changes affect the stroma and the tumor cells resulting in a microenvironment that prevents proliferation of breast tumors and their metastases.     

Targeting superoxide dismutase to renal proximal tubule cells inhibits nephrotoxicity of cisplatin and increases the survival of cancer-bearing mice

Because cis-diamminedichloroplatinum(II) (cisplatin) which generates reactive oxygen species induces renal dysfunction, administration of a large dose for killing cancer cells is highly limited. We recently synthesized a cationic superoxide dismutase (SOD) (hexamethylenediamine-conjugated SOD, AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. Treatment of Ehrlich ascites tumor cells (EATC)-bearing mice with cisplatin sufficient for killing tumor cells increased their motality. The motality of cisplatin-treated EATC-bearing mice was markedly decreased by AH-SOD. These results suggest that targeting SOD to renal proximal tubule cells might permit the administration of high doses of cisplatin and related anticancer agents without causing renal injury.     

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation

Background:

Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development.

Methods:

To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53^lox/lox/FAK^+/+/WapCre, p53^lox/lox/FAK^flox/+/WapCre and p53^lox/lox/FAK^flox/−/WapCre mice, and mice with WapCre-mediated conditional expression of p53^R270H, the mouse equivalent of human p53^R273H hot spot mutation, together with conditional deletion of FAK, P53^R270H/+/FAK^lox/+/WapCre and p53^R270H/+/FAK^flox/−/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours.

Results:

Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53^R270H-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures.

Conclusions:

Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53^R270H mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53^R270H-induced mammary tumour development.     

Protein tyrosine phosphatase epsilon increases the risk of mammary hyperplasia and mammary tumors in transgenic mice

Accurate phosphorylation of tyrosine residues in proteins plays a central role in regulation of cellular function. Although connections between aberrant tyrosine kinase activity and malignancy are well-established, significantly less is known about the roles of protein tyrosine phosphatases (PTPases) in tumorigenesis. We have previously shown that the transmembranal form of PTPase Epsilon (PTPepsilon) is upregulated in mouse mammary tumors initiated specifically by ras or neu, suggesting that PTPepsilon may play a role in transformation by these two oncogenes. In order to test this notion in vivo, we created transgenic mice that express elevated levels of PTPepsilon in their mammary epithelium by use of the MMTV promoter/enhancer. Following several cycles of pregnancy female MMTV-PTPepsilon mice uniformly developed pronounced and persistent mammary hyperplasia which was accompanied by residual milk production. Solitary mammary tumors were often detected secondary to mammary hyperplasia. The sporadic nature of the tumors, the long latency period prior to their development, and low levels of transgene expression in the tumors indicate that PTPepsilon provides a necessary, but insufficient, signal for oncogenesis. The results provide genetic evidence that PTPepsilon plays an accessory role in production of mammary tumors in a manner consistent with its upregulation in mammary tumors induced by ras or neu.     

Co-culture with human fibroblasts increases the radiosensitivity of MCF-7 mammary carcinoma cells in collagen gels

The growth and differentiation of normal and neoplastic epithelial cells may be regulated by the presence of adjacent normal tissues and cells, particularly stromal fibroblasts. However, the influence of normal fibroblast-tumor cell interactions on the response of malignant epithelial cells to radiation has not been adequately investigated nor has the possible role played by a 3-D environment in such modulation. We addressed this question by embedding MCF-7 mammary carcinoma cells into a collagen lattice, alone or mixed with HSF human dermal fibroblasts, and kept the gels anchored to the plastic surface or suspended in the culture medium. Some gels served as controls and others were irradiated with 6 MV photons fractionated into 3 daily doses totaling 5 or 10 Gy. After 2 or 7 days from the last treatment (7 or 12 days in culture, respectively), gels were processed in 1 of 2 ways: overall cell survival was determined by the MTT assay, while the survival of MCF-7 cells was selectively detected by a clonogenicity assay. Under these experimental conditions, we found that, in the presence of HSF fibroblasts, the growth of MCF-7 cells was restrained and radiosensitivity increased compared with MCF-7 cells cultured alone. For example, while the average number of MCF-7 foci/gel recovered from control gels with MCF-7 cells alone was 2,460 on day 7 and 3, 290 on day 12 of culture, it was 4 to 5 times lower (p < 0.001) in control gels with mixed MCF-7 and HSF cells. Radiation affected severely the survival of MCF-7 cells in all experimental groups but not sufficiently to mask the differences. For example, following exposure to the low dose of 5 Gy, the average number of MCF-7 foci/gel recovered from MCF-7-containing gels was 590 on day 7 and 329 on day 12 of culture, whereas numbers from the gels containing mixed MCF-7 and HSF cells were only 218 and 73, respectively (p < 0. 003 in both cases). HSF fibroblasts did not grow in our system, but they contracted strongly anchored and floating gels.     

In utero alcohol exposure increases mammary tumorigenesis in rats

Findings in humans and animal models suggest that in utero hormonal and dietary exposures increase later breast cancer risk. Since alcohol intake by adult women consistently increases their breast cancer risk, we wondered whether maternal alcohol consumption during pregnancy increases female offspring's mammary tumorigenesis. In our study, pregnant female rats were pair-fed isocaloric diets containing either 0 (control), 16 or 25 g alcohol kg(-1) feed between days 7 and 19 of gestation. These alcohol exposures generate blood alcohol levels that correspond to low and moderate alcohol consumption and are lower than those that induce foetal alcohol syndrome. Serum oestradiol levels were elevated in pregnant rats exposed to alcohol (P<0.003). When adult, female offspring of alcohol-exposed dams developed significantly more 7,12-dimethylbenz[a]anthracene -induced mammary tumours, compared to the controls (tumour multiplicity; mean+/-s.e.m., controls: 2.0+/-0.3, 16 g alcohol: 2.7+/-0.4 and 25 g alcohol: 3.7+/-0.4; P<0.006). In addition, the mammary epithelial tree of the alcohol-exposed offspring was denser (P<0.004) and contained more structures that are susceptible for the initiation of breast cancer (P<0.001). Immunohistochemical assessment indicated that the mammary glands of 22-week-old in utero alcohol-exposed rats contained elevated levels of oestrogen receptor-alpha (P<0.04) that is consistent with the changes in mammary gland morphology. In summary, maternal alcohol intake during pregnancy increases female offspring's mammary tumorigenesis, perhaps by programming the foetal mammary gland to exhibit persistent alterations in morphology and gene expression. It remains to be determined whether an increase in pregnancy oestradiol levels mediated alcohol's effects on offspring's mammary tumorigenesis.