A persistent lactation is dependent on maintaining the number and activity of milk secreting cells with advancing lactation. When dairy cows are milked twice daily, the increase in milk yield from parturition to peak lactation is due to increased secretory activity per cell rather than to accretion of additional epithelial cells. After peak lactation, declining milk yield is due to loss of mammary epithelial cells by apoptosis. During lactation, only 0.3% of mammary cells proliferate in a 24-h period. Yet this proliferative rate is sufficient to replace most mammary epithelial cells by the end of lactation. Management practices can influence lactation persistency. Administration of bovine somatotropin may enhance persistency by increasing cell proliferation and turnover, or by reducing the rate of apoptosis. Increased photoperiod may also increase persistency of lactation by mechanisms that are as yet undefined. Increased milking frequency during the first weeks of lactation increases milk yield, even after return to less frequent milking, with increases of approximately 8% over the entire lactation. A mammary cell proliferation response to frequent milking during early lactation appears to be involved. Conversely, advanced pregnancy, infrequent milking, and mastitis increase death of epithelial cells by apoptosis. Regulation of mammary cell renewal provides a key to increasing persistency. Investigations to characterize epithelial cells that serve as the proliferative population in the bovine mammary gland have been initiated. Epithelial cells that stain lightly in histological sections are evident through all phases of mammary development and secretion and account for nearly all proliferation in the prepubertal gland. Characterization of these cells may provide a means to regulate mammary cell proliferation and thus to enhance persistency, reduce the effects of mastitis, and decrease the necessity for a dry period.

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The thymus and parathyroid glands in mice develop from a thymus/parathyroid primordium that forms from the endoderm of the third pharyngeal pouch. We investigated the molecular mechanisms that promote this unique process in which two distinct organs form from a single primordium, using mice mutant for Hoxa3 and Pax1. Thymic ectopia in Hoxa3(+/-)Pax1(-/-) compound mutants is due to delayed separation of the thymus/parathyroid primordium from the pharynx. The primordium is hypoplastic at its formation, and has increased levels of apoptosis. The developing third pouch in Hoxa3(+/-)Pax1(-/-) compound mutants initiates normal expression of the parathyroid-specific Gcm2 and thymus-specific Foxn1 genes. However, Gcm2 expression is reduced at E11.5 in Pax1(-/-) single mutants, and further reduced or absent in Hoxa3(+/-)Pax1(-/-) compound mutants. Subsequent to organ-specific differentiation from the shared primordium, both the parathyroids and thymus developed defects. Parathyroids in compound mutants were smaller at their formation, and absent at later stages. Parathyroids were also reduced in Pax1(-/-) mutants, revealing a new function for Pax1 in parathyroid organogenesis. Thymic hypoplasia at later fetal stages in compound mutants was associated with increased death and decreased proliferation of thymic epithelial cells. Our results suggest that a Hoxa3-Pax1 genetic pathway is required for both epithelial cell growth and differentiation throughout thymus and parathyroid organogenesis.

BackgroundUnlike glycolytic enzymes that directly catabolize glucose to pyruvate, the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs) control the conversion of fructose-6-phosphate to and from fructose-2,6-bisphosphate, a key regulator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). One family member, PFKFB3, has been shown to be highly expressed and activated in human cancer cells, and derivatives of a PFKFB3 inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), are currently being developed in clinical trials. However, the effectiveness of drugs such as 3PO that target energetic pathways is limited by survival pathways that can be activated by reduced ATP and nutrient uptake. One such pathway is the process of cellular self-catabolism termed autophagy. We hypothesized that the functional glucose starvation induced by inhibition of PFKFB3 in tumor cells would induce autophagy as a pro-survival mechanism and that inhibitors of autophagy could increase the anti-tumor effects of PFKFB3 inhibitors.ResultsWe found that selective inhibition of PFKFB3 with either siRNA transfection or 3PO in HCT-116 colon adenocarcinoma cells caused a marked decrease in glucose uptake simultaneously with an increase in autophagy based on LC3-II and p62 protein expression, acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. The induction of autophagy caused by PFKFB3 inhibition required an increase in reactive oxygen species since N-acetyl-cysteine blocked both the conversion of LC3-I to LC3-II and the increase in acridine orange fluorescence in acidic vesicles after exposure of HCT-116 cells to 3PO. We speculated that the induction of autophagy might protect cells from the pro-apoptotic effects of 3PO and found that agents that disrupt autophagy, including chloroquine, increased 3PO-induced apoptosis as measured by double staining with Annexin V and propidium iodide in both HCT-116 cells and Lewis lung carcinoma (LLC) cells. Chloroquine also increased the anti-growth effect of 3PO against LLCs in vivo and resulted in an increase in apoptotic cells within the tumors.ConclusionsWe conclude that PFKFB3 inhibitors suppress glucose uptake, which in turn causes an increase in autophagy. The addition of selective inhibitors of autophagy to 3PO and its more potent derivatives may prove useful as rational combinations for the treatment of cancer.

Exposure to short day photoperiod (SD; 8 h light:16 h dark) during the dry period increased milk yield of cows in the subsequent lactation. We hypothesized that this effect is due to increased growth of mammary cells in response to enhanced prolactin signaling to influence the insulin-like growth factor (IGF) axis. Multiparous Holstein cows were dried off 60 d before parturition and assigned to long day photoperiod (LD; 16 h light:8 h dark) or SD during the dry period. Mammary biopsies were obtained at approximately -40, -20, -10 and +10 d relative to expected calving. Expression of IGF-I, IGF-II, and IGF binding protein-5 mRNA was assessed by real time reverse transcription-polymerase chain reaction. In SD cows, incorporation of [3H]-thymidine in vitro increased from -40 d to -20 d and was greater at -20 d than in LD cows. A later increase in proliferation was observed at -10 d in LD cows. For both groups, cell proliferation decreased during lactation. Analysis by terminal deoxynucleotidyl transferase dUTP nick end labeling revealed that the apoptotic index in mammary epithelial cells was less in SD cows than in LD cows. Expression of IGF-II mRNA increased during the dry period and into lactation and was greater in SD cows. Expression of IGF binding protein-5 mRNA increased during lactation, but was unaffected by day length. Expression of IGF-I did not differ over time or between treatments. We concluded that exposure to SD during the dry period enhanced mammary growth relative to LD, and this may be related to increased expression of IGF-II. Treatment differences in the temporal pattern of proliferation indicated the existence of a critical period wherein photoperiod affects mammary gland development during the dry period.