Bovine mammary epithelial (MAC-T) cells are commonly used to study mammary gland development and mastitis. Lipopolysaccharide is a major bacterial cell membrane component that can induce inflammation. Autophagy is an important regulatory mechanism participating in the elimination of invading pathogens. In this study, we evaluated the mechanism underlying bacterial mastitis and mammary cell death following lipopolysaccharide treatment. After 24 h of 50 μg/mL lipopolysaccharide treatment, a significant decrease in the proliferation rate of MAC-T cells was observed. However, no changes were observed upon treatment of MAC-T cells with 10 μg/mL of lipopolysaccharide for up to 48 h. Thus, upon lipopolysaccharide treatment, MAC-T cells exhibit dose-dependent effects of growth inhibition at 10 μg/mL and death at 50 μg/mL. Treatment of MAC-T cells with 50 μg/mL lipopolysaccharide also induced the expression of autophagy-related genes ATG3, ATG5, ATG10, ATG12, MAP1LC3B, GABARAP-L2, and BECN1. The autophagy-related LC3A/B protein was also expressed in a dose-dependent manner upon lipopolysaccharide treatment. Based on these results, we suggest that a high dose of bacterial infection induces mammary epithelial cell death related to autophagy signals.

Natural killer (NK) cells have cytotoxic effects on tumor cells and viral pathogens. NK cell-derived exosomes (NK-exosomes) also express typical NK cell markers and cytotoxic molecules, therefore, exert anti-tumor and immune homeostatic activities. In this study, canine NK-exosomes separated from cytotoxic NK cell supernatant carried specific markers such as CD81, Alix, and Perforin 1. We examined the anti-tumor effects of NK-exosomes in an experimental murine model using the canine mammary carcinoma cells, REM134. REM134 cells were xenografted of mammary fat pad of mice. CD133, Bmi-1, MMP-3, IL-6, TNF-α, and PCNA are useful as a molecular marker for tumorigenesis and metastasis. The treatment of canine NK-exosomes inhibited tumor growth and significantly (p<0.01) downregulated the expression of Bmi-1, MMP-3, IL-6, TNF-α, and PCNA in REM134-treated mice. Also, the expression of CD133, potent cancer stem cell marker, was significantly downregulated in the canine NK-exosomes-treated mice compared with that of the tumor group. Collectively, these results suggested that canine NK-exosomes has a potential capacity for regulation of cancer progression and metastasis against canine mammary carcinoma.

Canine mammary tumors account for ~30% of all tumors in the female dogs and approximately 50% of the tumors are malignant. Exosomes have been the focus of great interest, as they appear to be involved in numerous important cellular processes. In this study, we examined the anti-tumor effects of canine mesenchymal stem cells-derived exosomes (MSC-exosomes) in an experimental murine mammary tumor model using canine mammary carcinoma cells, REM134. The MSC-exosomes were injected tumor site and tail vein of REM134 xenografted mice. We found that tumor size of the MSC-exosomes-treated group decreased compared to those of the only tumor group in REM134-driven tumorigenic mouse model. In addition, the MSC-exosomes-treated tumor group showed meaningfully reduced expression levels of the MMP-3, IL-1β, IL-6, and TNF-α compared to those in the tumor group. Specifically, we confirmed that the expression level of the CD133, potent cancer stem cell (CSC) markers, decreased in the MSC-exosomes-treated tumor group compared to the tumor group. This study suggests that the MSC-exosomes exhibited anti-tumor effects through downregulating CSC-related markers in the canine mammary tumor murine model. Further study is needed in the future, and we are conducting research on the detailed anti-tumor mechanism of the MSC-exosomes.

Canine inflammatory mammary carcinoma (CIMC) similar to human inflammatory breast cancer is a very aggressive, metastatic type of cancer. Previous studies have introduced a new type of tumor angiogenesis called vasculogenic mimicry that may play an important role in the progression of inflammatory mammary cancer. The purpose of this study is to investigate the development process of vessels by neoplastic cells in CIMC. Patient dog, 14-year old Shit-Tzu female, had a hard and somewhat movable dark-reddish mammary tumor, sized 6.2 cm in diameter. Bloody dark turbid exudate was released from the tumor. In addition to histological examination, immunohistochemistry for pancytokeratin, VCAM-1, MECA- 32, TWIST-1, and Ki-67 was respectively performed using the ABC method. Histologically, the inflammatory mammary carcinoma was characterized by tubular solid tumor emboli within the lymphatic vessels surrounded by desmoplastic fibrous connective tissue. Some of the neoplastic cells were transforming into elongate or spindle shapes and forming small vessel-like structures in the solid tumor mass. The neoplastic cells were immunoreactive for VCAM-1 and MECA-32, but showed low immunoreactivity for Ki-67. Immunoreactive neoplastic cells for VCAM-1 and MECA-32 suggested the possibility that the neoplastic cells transform into endothelial cells of vessels by epithelial-mesenchymal transition, further supported by serial morphological changes identified by histological investigation and immunohistochemistry for TWIST-1. The high capacity of the neoplastic cells forming the vasculatures in CIMC explains the high ratio of metastasis to other regions, even though Ki-67 index was not so high.

The mammary gland tumor (MGT) is the most common neoplasia in intact female dogs. Of these, 50% are malignant and metastasis to the other sites are often occurred. Therefore, it is very important for decision of treatment plan and prognosis to differentiate benign tumor from malignancies. Calcification of MGT is a very important imaging finding. The purpose of this study was to investigate the radiological and computed tomographic images of the MGT and the morphology and distribution of calcifications in the MGT using the Breast Imaging Reporting and Data System classification. A total of 42 dogs with MGT were included in this study. The dogs were divided into two groups into benign and malignant groups based upon histologic or cytologic results. The appearance of calcification in the tumor on radiographs and CT images was analyzed for the HU value of pre- and post-contrast injection, margin, surface, and shape of the tumor and the lymph node abnormalities. On radiographs, the positive predictive value of malignant and benign tumors was 72.72 and 85.71%, respectively. On CT examinations, the positive predictive value of malignant and benign tumors was the same value of 83.33%. The maximum diameter of the tumor and the presence of abnormal lymph nodes on CT images showed a strong correlation with malignancies. Therefore, it is thought that radiographs and CT provide useful information for evaluating MGT in dogs.

The mammary gland is a complex organ, made up of various cell types that work together for mil synthesis. A previous study had established a clonal cell line from primary bovine mammary alveolar cells (MAC-T) for the study of bovine milk production and synthesis. In this study, we transplanted MAC-T bovine alveolar cell line to BALB/C nude mice for regeneration of bovine mammary gland alveolar ducts. The MAC-T cells were suspended with matrigel and injected into the dorsal tissue of 8 weeks old male BALB/C nude mice. After 6 weeks, the injected cells were showed typical morphology of the tubuloalveolar female glands by histological analysis. It was made the branching ducts that were surrounded by smooth muscle with small alveoli budding off the ducts. In addition, the epithelial markers CK14 and CK18 were expressed within the duct-like structure. Prolactin was detected in the duct interior in these CK14 and CK18 positive cells but not in the non-transplanted MAC-T cells. These results showed that duct-like tissue had been successfully formed after 6 weeks of transplantation of the CK14 and CK18 positive MAC-T cells into mice dorsal tissue. This mouse model will be a useful tool for further research on the bovine mammary gland.

Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat β-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.

The objective of the research was to identify the presence of adiponectin receptors and to study adiponectin action on glucose uptake and growth in mouse mammary epithelial cells. These cells expressed adiponectin receptors, AdipoR1 and AdipoR2. Insulin (10 ng/ml) or insulin like growth factor-I (IGF-I, 10 ng/ml) alone did not alter the degree of AdipoR1 and AdipoR2 genes expression from 0 to 4 h incubation. Prolactin (10 ng/ml) or epidermal growth factor (EGF, 10 ng/ml) alone also did not induce the two genes’ mRNA in the incubation time. Adiponectin (1 μg/ml) alone or pre-incubation of insulin alone (100 ng/ml) for 2 h prior to adiponectin stimulation did not increase 2-deoxy-D-glucose,[1,2-3H] uptake but adiponectin+pre-incubation of insulin significantly increased glucose uptake compare to control (p<0.05). In a similar way, insulin alone or pre-incubation of adiponectin alone (2 h) did not increase glucose uptake but insulin+pre-incubation of adiponectin increased glucose uptake compare to control (p<0.05). Insulin sensitization for 2 h prior to adiponectin stimulation tended to increase glucose uptake response by the following adiponectin stimulation showing small interaction effect between insulin and adiponectin (p<0.1). However, adiponectin sensitization for 2 hours prior to insulin stimulation did not shown interaction effect between adiponectin and insulin (p>0.1). The glucose uptake by both of hormones seems to be not interactive but additive (p<0.05). Adiponectin in the presence of 2% FBS decreased DNA synthesis of mammary epithelia (p<0.05). AICAR (100 or 200 μM), AMPK activator, decreased mammary epithelial cell growth in the presence of 2% FBS. These results indicate that adiponectin pathway has inhibitory effect on mammary epithelial cell growth.

형질 전환 동물 생산에는 조직 및 시기 특이적 발현 조절이 가능하다는 장점 때문에 유즙 내로 외부 유전자를 발현시키는 시스템이 널리 이용되고 있다. 유전자 발현 즉, 단백질 생산은 프로모터의 강도뿐만 아니라 mRNA의 안정성에 의해서도 조절된다. 특히, polyadenylation에 의한 poly A의 길이는 in vivo와 올 in vitro에서 mRNA 안정성 및 목적 유전자의 번역효율에 영향을 준다. 본 연구에서는 이러한 mRNA 안정성이 목적 유전자의 발현에 미치는 영향을 알아보기 위해 3'-UTR 염기 서열을 분석하였다. 이 3'-untranslated region(UTR) 내의 poly A signal을 기준으로 putative cytoplasmic polyadenylation element(CPE) 부위와 downstream elements(DSE: U-rich, G-rich, GU-rich)의 염기 서열을 분석하고, 각각의 element를 기준으로 15 종의 luciferase reporter vector를 제작하여, 생쥐 유선 세포주(HC11)와 돼지 유선 세포주(PMGC)에 각각 transfection시킨 후 48시간 동안 배양하고 luciferase 발현량을 분석하였다. PMGC의 경우, luciferase의 발현은 exon 9의 CPE 2,3 및 DSE 1을 포함한 #6 construct에서 유의적으로 높은 발현량을 보였으며, exon 9의 CPE 2, 3과 DSE를 모두 포함하고 있는 #11 construct에서도 유의적으로 높은 발현량을 보였다. 이러한 결과는 형질 전환 돼지 생산에 있어 #6 및 11 construct의 사용은 목적의 유전자를 효과적으로 발현시키는데 기여할 것으로 사료된다.

The effects of adenosine 5′-triphosphate (ATP) and ATP analogs, P/sub 2y/ purinoceptor agonists, on growth of normal mouse mammary epithelial cells (NMuMG) were examined. Cells were plated onto 24 well plates in DMEM supplemented with 10 % fetal calf serum. After serum starvation for 24 hours, ATP, P/sub 2y/ purinoceptor agonists (AdoPP[NH]P, ATP-α-S, ATP-γ-S, β, γ-me-ATP and 2me-S-ATP), P/sub 2u/ purinoceptor agonist (UTP) and P/sub 2y/ purinoceptor antagonists (Reactive Blue 2, more selective to P/sub 2y/ receptor than PPADS; PPADS) were added. DNA synthesis was estimated as incorporation of 3H-thymidine into DNA (1 hour pulse with 1 μ Ci/ml, 18～19 hours after treatment). ATP, Adopp[NH]P, ATP-α-S or ATP-γ-S, significantly increased DNA synthesis at 1, 10 and 100 μM concentrations with dose-dependency (P<0.05), and the maximum responses of ATP and ATP analogs were shown at 100 μM concentration (P<0.05). The potency order of DNA synthesis was ATP≥ATP- γ -S>Adopp [NH]P>ATP-α-S. β, γ -me-ATP, 2me-S-ATP and UTP did not increase DNA synthesis. In autoradiographic analysis of percentage of S-phase cells, similar results were observed to those of DNA synthesis. Addition of 1, 10 or 100 μM Reactive Blue 2 or PPADS significantly decreased ATP (100 μM)-induced DNA synthesis, however, PPADS was less effective than Reactive Blue 2. In Elvax 40P implant experiment, ATP directly stimulated mammary endbud growth in situ suggesting the physiological regulator of ATP in mammary growth. ATP 100 μM rapidly increased MAPK activity, reaching a maximum at 5 min and then gradually decreasing to the base level in 30 min. ATP analogs, Adopp[NH]P and ATP-γ-S also increased MAPK activity, however, β, γ-me-ATP and 2me-S-ATP did not. The inhibitor of the upstream MAPK kinase (MEK), PD 98059 (25 μM), effectively reduced ATP (100 μM) or EGF(10 ng/ml, as positive control)-induced MAPK activity and DNA synthesis (P<0.05). These results indicate that ATP-induced DNA synthesis was prevented from the direct inhibition of MAPK kinase pathway. Overall results support the hypothesis that the stimulatory effects of normal mouse mammary epithelial growth by addition of ATP or ATP analogs are mediated through mammary tissue specific P/sub 2y/ purinoceptor subtype, and MAPK activation is necessary for the ATP-induced cell growth.

본 연구는 VSV-G glycoprotein을 envelope으로 하는 pseudotyped retrovirus vector system을 이용하여 쥐의 유방상피세포인 HC11에서 human Lactadherin 유전자의 발현을 확인하고자 하였다. 실험에 사용한 vector는 개체내에서의 외래 유전자의 지속적인 발현에 의한 생리적인 부작용을 최소화하기 위한 구조로, 조직특이적이며 lactogenic hormone에 의해 유도적인 활성을 가지는 것으로 알려진 WAP promoter의 통제하에 도입하고자 하는 외래 유전자를 위치하도록 하였다. WAP promoter의 대조군으로 지속적인 활성을 나타내는 β-actin promoter를 사용하였으며, 이 각각의 promoter와 marker gene으로 E. coli LacZ gene을 재조합한 후 retrovirus vector system을 이용하여 HCll에 도입하였다. 세포의 genome 내로의 유전자의 전이는 PCR을 통해 확인하였고, RT-PCR의 수행으로 유전자의 발현을 확인하였다. Lactadherin 유전자를 이용한 실험도 동일한 과정으로 수행하였으며, RT-PCR의 결과에서 HCll 세포에서 Lactadherin 유전자의 발현이 insulin을 단독으로 처리한 군에 비해 insulin, hydrocortisone, prolactin을 동시에 처리한 군에서 우월하게 나타나는 것으로 확인되었다. 그러나 insulin 단독 처리군에서 유전자의 발현이 약하게 나타나는 것으로 관찰되어 WAP promoter의 leakiness에 대한 재고의 필요성이 요구되었다.

Glucocorticoid는 비유기 동물의 유선세포 pro-lactin receptor(PRL-R) 발현을 증가시키며, 전반적인 유선세포의 유합성 작용을 활성시킴으로 유합성 능력을 증진시킨다. 유선세포 PRL-R 발현량 증가는 유생산량 향상과 밀접한 관계를 갖는다. 본 실험은 비유중기의 재래산양의 유합성 능력을 향상시키고자, 0.05. 0.1과 0.2 g hydrocortisone을 5ml의 생리식염수에 현탁하여, 정맥투여하고 유선세포 PRL-R와 -유

Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinomas and may prevent symptomatic rotavirus infections. In this study, under the control of mouse whey acidic protein (WAP) promoter, the expression pattern of lactadherin (Ltd) in lactogenic hormone-dependent mouse mammary epithelial cell line HC11 were tested. pLNWLtd construct containing 2.4 kilobases of the WAP promoter and 1.5 kilobases of human lactadherin gene was stably transfered into HC11 cells using retroviral vector system. Integration and expression level of the transgene was estimated using PCR and RT-PCR, respectively. Prominent induction of Ltd gene under the WAS promoter was accomplished in the presence of insulin, hydrocortisone and prolactin, while induction with insulin alone resulted in lower expression. Our results demonstrate that the expression of the transgene is increased by synergistic effect of several lactogenic hormones, including insulin, hydrocortisone, and prolactin.