Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. (C) 2003 Wiley Periodicals, Inc.

MMTV-trBrca 1 mice display strain-dependent abnormalities in vaginal development

The breast cancer susceptibility gene Brca1 encodes a large multi-functional protein which is implicated as a caretaker of the genome, through its role in regulation of DNA damage response pathways, including apoptosis. Here we show that in mice expressing a dominant-negative Brca1 transgene on a BALB/c background, vaginal entrance remodeling is inhibited, and that the incidence of this phenotype is increased on a p53 +/- genotype. Given that this developmental process is mediated primarily by apoptosis, we hypothesized that disruption of BRCA1 may confer a resistance to apoptosis in normal epithelial cells. Consistent with this, we show that expression of this transgene in vitro leads to resistance to ionizing radiation induced cell killing in mammary epithelial cells. This is the first time that BRCA1 has been implicated in an apoptosis-mediated normal developmental process.

Chronic renal failure in an English bull terrier with polycystic kidney disease

An entire female English bull terrier, aged five years and one month, was diagnosed with polycystic kidney disease by renal ultrasonography. It had thickening and abnormal motion of the mitral valve on 2D and M mode echocardiography, and left ventricular outflow tract obstruction, characterised by turbulence in the left ventricular outflow tract and elevated aortic blood flow velocity, detected by colour flow and spectral Doppler echocardiography, respectively. Two years later, haematology, serum biochemistry and urinalysis data suggested the presence of compensated renal failure. The dog was euthanased at 10 years and eight months of age, with haematology, serum biochemistry and urinalysis data indicating decompensated chronic renal failure. Postmortem examination confirmed polycystic kidney disease, chronic renal disease, mitral and aortic valvular myxomatous degeneration, and mixed mammary neoplasia. This case demonstrates that bull terriers with polycystic kidney disease may develop associated chronic renal failure.

Pseudopregnancy in the bitch

Pseudopregnancy in the bitch is a normal phenomenon. It refers to the dioestrus phase of the bitch's reproductive cycle. It can range in severity from covert pseudopregnancy, in which the signs are barely noticeable, to severe clinical or overt pseudopregnancy. It occurs six to eight weeks following oestrous. The bitch may present with pregnancy-like behaviour including nesting and aggression, excessive mammary enlargement and contractions. These signs are an exaggerated version of the normal signs shown in a normal entire cycling bitch. The exact aetiology behind pseudopregnancy is not known. It is believed to be associated with a rapid decline in serum progesterone concentrations and the resulting surge in prolactin. Treatment may involve conservative management, medical therapy or ovariectomy/ovariohysterectomy. In the past medical therapy has included sex steroids including oestrogens, androgens and progestins. However, due to the frequent incidence and severity of side effects their use is contraindicated. Recommended forms of management incorporate the use of prolactin inhibitors such as cabergoline, metergoline and bromocriptine. These drugs reduce serum levels of prolactin and therefore reduce the development of mammary glands and decrease the behavioural signs associated with overt pseudopregnancy.

Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines

There is evidence to suggest that plasma membrane Ca2+-ATPase (PMCA) isoforms are important mediators of mammary gland physiology. PMCA2 in particular is upregulated extensively during lactation. Expression of other isoforms such as PMCA4 may influence mammary gland epithelial cell proliferation and aberrant regulation of PMCA isoform expression may lead or contribute to mammary gland pathophysiology in the form of breast cancers. To explore whether PMCA2 and PMCA4 expression may be deregulated in breast cancer, we compared mRNA expression of these PMCA isoforms in tumorigenic and non-tumorigenic human breast epithelial cell lines using real time RT-PCR. PMCA2 mRNA has a higher level of expression in some breast cancer cell lines and is overexpressed more than 100-fold in ZR-75-1 cells, compared to non-tumorigenic 184135 cells. Although differences in PMCA4 mRNA levels were observed between breast cell lines, they were not of the magnitude observed for PMCA2. We conclude that PMCA2 mRNA can be highly overexpressed in some breast cancer cells. The significance of PMCA2 overexpression on tumorigenicity and its possible correlation with other properties such as invasiveness requires further study. (c) 2005 Elsevier Inc. All rights reserved.

Antisense-mediated inhibition of the plasma membrane Calcium-ATPase suppresses proliferation of MCF-7 cells

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anticancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

An immunomodulator used to protect young in the pouch of the Tammar wallaby, Macropus eugenii

Eugenin [pGluGlnAspTyr(SO3)ValPheMetHisProPhe-NH2] has been isolated from the pouches of female Tammar wallabies (Macropus eugenii) carrying young in the early lactation period. The sequence of eugenin has been determined using a combination of positive and negative ion electrospray mass spectrometry. This compound bears some structural resemblance to the mammalian neuropeptide cholecystokinin 8 [AspTyr(SO3)MetGlyTrpMetAspPhe-NH2] and to the amphibian caerulein peptides [caerulein: pGluGlnAspTyr(SO3)ThrGlyTrpMetAspPhe-NH2]. Eugenin has been synthesized by a route which causes only minor hydrolysis of the sulfate group when the peptide is removed from the resin support. Biological activity tests with eugenin indicate that it contracts smooth muscle at a concentration of 10(-9) m, and enhances the proliferation of splenocytes at 10(-7) M, probably via activation of CCK2 receptors. The activity of eugenin on splenocytes suggests that it is an immunomodulator peptide which plays a role in the protection of pouch young.

Promoter-, cell-, and ligand-specific transactivation responses of the VDRB1 isoform

The vitamin D receptor (VDR) mediates the effects of 1,25(OH)(2)D-3, the active form of vitamin D. The human VDRB1 isoform differs from the originally described VDR by an N-terminal extension of 50 amino acids. Here we investigate cell-, promoter-, and ligand-specific transactivation by the VDRB1 isoform. Transactivation by these isoforms of the cytochrome P450 CYP24 promoter was compared in kidney (HEK293 and COS1), tumor-derived colon (Caco-2, LS174T, and HCT15), and mammary (HS578T and MCF7) cell lines. VDRB1 transactivation in response to 1,25(OH)(2)D-3 was greater in Cost and HCT15 cells (145%), lower in HEK293 and Caco-2 cells (70-85%) and similar in other cell lines tested. By contrast, on the cytochrome P450 CYP3A4 promoter, 1,25(OH)(2)D-3-induced VDRB1 transactivation was significantly lower than VDRA in Caco-2 (68%), but comparable to VDRA in HEK293 and COS1 cells. Ligand-dependence of VDRB1 differential transactivation was investigated using the secondary bile acid lithocholic acid (LCA). On the CYP24 promoter LCA-induced transactivation was similar for both isoforms in COS1, whereas in Caco-2 and HEK293 cells VDRB1 was less active. On the CYP3A4 promoter, LCA activation of VDRB1 was comparable to VDRA in all the cell lines tested. Mutational analysis indicated that both the 1,25(OH)(2)D-3 and LCA-regulated activities of both VDR isoforms required a functional ligand-dependent activation function (AF-2) domain. In gel shift assays VDR:DNA complex formation was stronger in the presence of 1,25(OH)(2)D-3 than with LCA. These results indicate that regulation of VDRB1 transactivation activity is dependent on cellular context, promoter, and the nature of the ligand. (c) 2005 Elsevier Inc. All rights reserved.