Sex- and age-dependent effects of androgens on glutamate-induced cell death and intracellular calcium regulation in the developing hippocampus.

Hippocampal neurons must maintain control over cytosolic calcium levels, especially during development, as excitation and calcium flux are necessary for proper growth and function. But excessive calcium can lead to excitotoxic cell death. Previous work suggests that neonatal male and female hippocampal neurons regulate cytosolic calcium differently, thereby leading to differential susceptibility to excitotoxic damage. Hippocampal neurons are also exposed to gonadal hormones during development and express high levels of androgen receptors. Androgens have both neuroprotective and neurotoxic effects in adults and developing animals. The present study sought to examine the effect of androgen on cell survival after an excitatory stimulus in the developing hippocampus, and whether androgen-mediated calcium regulation was the governing mechanism. We observed that glutamate did not induce robust or sexually dimorphic apoptosis in cultured hippocampal neurons at an early neonatal time point, but did 5days later - only in males. Further, pretreatment with the androgen dihydrotestosterone (DHT) protected males from apoptosis during this time, but had no effect on females. Calcium imaging of sex-specific cultures revealed that DHT decreased the peak of intracellular calcium induced by glutamate, but only in males. To determine a possible mechanism for this androgen neuroprotection and calcium regulation, we quantified three calcium regulatory proteins, plasma membrane calcium ATPase1 (PMCA1), sodium/calcium exchanger1 (NCX1), and the sarco/endoplasmic reticulum calcium ATPase 2 (SERCA2). Surprisingly, there was no sex difference in the level of any of the three proteins. Treatment with DHT significantly decreased PMCA1 and NCX1, but increased SERCA2 protein levels in very young animals but not at a later timepoint. Taken together, these data suggest a complex interaction of sex, hormones, calcium regulation and developmental age; however androgens acting during the first week of life are implicated in regulation of hippocampal cell death and may be an underlying mechanism for sexually dimorphic apoptosis.

A CD44 survival pathway triggers chemoresistance via lyn kinase and phosphoinositide 3-kinase/Akt in colon carcinoma cells.

A major obstacle to successful treatment of colorectal cancer is chemotherapy resistance. Enhanced expression of variant CD44 isoforms has been associated with aggressive tumor behavior, prompting the question of whether signaling from this receptor might modulate drug sensitivity. Activation of variant CD44 in colon carcinoma cell lines triggered resistance to the drug 1,3-bis(2-chloroethyl)-1-nitrosurea. Resistance was induced by monoclonal antibodies directed against epitopes independent of the hyaluronate-binding region but was not triggered by identical treatment of a carcinoma line expressing the standard CD44 isoform. We observed that variant CD44 produced activation of the src-family tyrosine kinase lyn. Moreover, overexpression of dominant-active lyn recapitulated chemoresistance via a pathway shown to involve activation of phosphoinositide 3-kinase and Akt. These results establish a novel role for CD44 in determining survival of colon carcinoma cells through lyn kinase and Akt. The ability to suppress apoptosis might play a critical role in the onset and development of colorectal malignancies.

Spheroids and cell survival.

In contrast to most traditional cell culture systems, spheroids represent a unique opportunity to recapitulate aspects of cell homeostasis and as such better reflect in vivo tumor biology. This review highlights recent spheroid-based studies which have defined a role for intercellular adhesion in dictating cellular fate. Critical roles for adhesion molecules, such as the integrin and cadherin families, have been established. In addition, it appears that components of the signaling pathways that control cellular proliferation may also influence the cellular decision to live or die. Further complexity arises from the action of soluble factors, like epidermal growth factor, and three-dimensional culture systems are providing insights into how integration of these multiple signals is achieved. Given that cellular adhesion may serve as a regulator of apoptosis, and in particular that correct receptor engagement can activate signaling pathways promoting cell survival, additional implications for our understanding of tumor biology are discussed.

Side-chain hydroxylation of vitamin D3 and its physiological implications.

Evidence is accumulating that, in vivo and in vitro, both 25-OH-D3 and 1,25-(OH)2D3 undergo side-chain modification leading to side-chain cleaved metabolites lacking the 24, 25, 26, and 27 carbons. The enzymes involved are D-dependent and are located in the kidney, bone, intestine, and perhaps other sites. We speculate that the extra-renal side-chain pathway may be primarily for target organ destruction of 1,25-(OH)2D3, whereas the renal pathway may be primarily for destruction of 25-OH-D3 formed in large amounts in hypervitaminosis D.

A time study comparing the appearance of catabolites of 9 alpha, 11 alpha, 15(S)-trihydroxyprosta-5,13-dienoic acid in blood and urine during constant intravenous infusion of tritiated 9 alpha, 11 alpha, 15(S)-trihydroxyprosta-5,13-dienoic acid in the rat. Isolation of a new circulating catabolite.

Profiles of 9 alpha, 11 alpha, 15(S)-trihydroxyprosta-5,13-dienoic acid (PGF2 alpha) and its catabolites in blood and urine of male adult Wistar rats were determined by high pressure liquid chromatography during and after slow intravenous infusion of tritium-labeled PGF2 alpha. In addition to PGF2 alpha and 15-keto-13,14-dihydro-PGF2 alpha, a major catabolite in blood during the infusion was identified as tetranor-15-keto-13,14-dihyro-PGF2 alpha (VII). This product was absent in the corresponding urine samples. VII was still detected in blood 2 h after the infusion was terminated. VII was previously shown by us to be an intermediate in the formation of certain urinary products of PGF2 alpha. These other catabolites of PGF2 alpha were also observed in both blood and urine although their levels were greater in the urine. Our observations indicate that a long lasting catabolite of PGF2 alpha, i.e. VII, as well as all the urinary catabolites of PGF2 alpha appear in the circulation. These results suggest that these catabolites are mostly (although not exclusively) extrarenal in origin (probably all hepatic) entering the circulation prior to their excretion by the kidney.

Identification of a 'urinary-type' metabolite of prostaglandin F2 alpha in the rat circulation.

During constant slow intravenous infusion of tritiated prostaglandin F2 alpha into male adult rats, a major portion of radioactivity in blood appeared as a new metabolite, identified as tetranor-15-keto-13,14-dihydroprostaglandin F2 alpha (18.8 +/- 4.2%, n = 7). The previously recognised blood metabolite, 15-keto-13,14-dihydroprostaglandin F2 alpha, was also observed (15.1 +/- 4.1%, n = 7). 15-keto-13,14-Dihydroprostaglandin F2 alpha disappeared quickly from the circulation while tetranor-15-keto-13,14-dihydroprostaglandin F2 alpha was still detected (8.6 +/- 2.8%, n = 3) 2 h after infusion was stopped. These results indicate that tetranor-15-keto-13,14-dihydroprostaglandin F2 alpha, because of its slow disappearance from the circulation, may provide a better indicator than 15-keto-13,14-dihydroprostaglandin F2 alpha of prostaglandin F2 alpha synthesis in vivo.

Distinct age-related changes in the urinary catabolic profile of prostaglandin F2 alpha in the rat.

Seven urinary catabolites were identified after intravenous infusion of tritium-labeled prostaglandin F2 alpha in the adult rat. Most products were the result of oxidation at C-15, reduction of the delta 13,14 double bond, and double beta oxidation together with omega oxidation reactions yielding 14 and 16 carbon compounds. While the former sequence of reactions is also demonstrable in the neonate (2 weeks and older), the omega oxidation system develops around 28 to 32 days of age. Consequently, the urinary catabolic pattern resulting from infusion of tritiated prostaglandin F2, in the rat younger than 32 days is composed principally of one product identified as 15-keto-13,14-dihydrotetranor prostaglandin F2 alpha (VII). This product is one of the transient intermediates in the adult and is catabolized further by omega oxidation.

Age-related differences in the urinary excretion of prostaglandin F2 alpha catabolites in the rat.

Tritium-labeled PGF2 alpha was administered i.v. into rats of varying ages (2, 4, 6 weeks and adult). Urine was collected and assayed for radioactive products by thin-layer-chromatography. Results showed a distinctly different urinary profile between the 2-week-old and the adult rat. While the urinary pattern from the 2-week-old rat gave a single less polar product than PGF2 alpha, the pattern from the adult rat gave products more polar than PGF2 alpha. Urine from the 4- and 6-week-old rats gave a mixture of these types of products. These results indicate that some prostaglandin catabolic pathway (likely the omega-oxidative system) is activated in vivo within the 4-6 week postnatal period in the rat.