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CA125 is a mucin commonly employed as a diagnostic marker for epithelial ovarian cancer. Induction of humoral responses to CA125 leads to increased survival times in patients with this form of cancer, suggesting a potential role for this mucin in tumor progression. In this study, oligosaccharides linked to CA125 derived from the human ovarian tumor cell line OVCAR-3 were subjected to rigorous biophysical analysis. Sequencing of the Oglycans indicates the presence of both core type 1 and type 2 glycans. An unusual feature is the expression of branched core 1 antennae in the core type 2 glycans. CA125 is also N-glycosylated, expressing primarily high mannose and complex bisecting type N-linked glycans. High mannose type glycans include Man 5 -Man 9 GlcNAc 2 . The predominant N-glycans are the biantennary, triantennary, and tetraantennary bisecting type oligosaccharides. Remarkably, the N-glycosylation profiles of CA125 and the envelope glycoprotein gp120 (derived from H9 lymphoblastoid cells chronically infected with HIV-1) are very similar. The CA125-associated N-glycans have also recently been implicated in crucial recognition events involved in both the innate and adaptive arms of the cell-mediated immune response. CA125 may therefore induce specific immunomodulatory effects by employing its carbohydrate sequences as functional groups, thereby promoting tumor progression. Immunotherapy directed against CA125 may attenuate these immunosuppressive effects, leading to the prolonged survival of patients with this extremely serious form of cancer.

Upon cell stimulation with hormones and other mitogens, a variety of biochemical and physiological responses occur within the first few minutes. Changes in both intracellular pH (pHin) and intracellular Ca2+ concentration ([Ca2+]in) are prominent and play a major role in the signal transduction mechanism leading to the physiological response, i.e., secretion, neurotransmission, proliferation, or differentiation. However, it is not clear whether these ions work independently in the activation pathway leading to a particular physiological response. The fluorescence characteristics of most Ca2+ indicators are pH sensitive, and quantitative estimates of [Ca2+]in cannot be made without knowledge of pHin. Thus it is desirable to have a technique to simultaneously monitor these two ions with relatively high time resolution. Here we have developed experimental conditions that allow us to use optimum emission conditions for a pH fluorescent indicator SNARF-1 and optimum excitation conditions for the Ca2+ indicator fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca2+ both in vitro and in situ. We have observed simultaneous changes in both pHin and [Ca2+]in in BALB/c 3T3 cells on treatment with the nonfluorescent Ca2+ ionophore 4-bromo-A23187. This temporal relationship between pHin and Ca2+ gives further credence to the interrelationship between these two second messengers in the expression of physiological responses.

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