Richard H. Knop

Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO2 or pH

Accumulation of CO(2) in animal cell cultures can be a significant problem during scale-up and production of recombinant glycoprotein biopharmaceuticals. By examining the cell-surface polysialic acid (PSA) content, we show that elevated CO(2) partial pressure (pCO(2)) can alter protein glycosylation. PSA is a high-molecular-weight polymer attached to several complex N-linked oligosaccharides on the neural cell adhesion molecule (NCAM), so that small changes in either core glycosylation or in polysialylation are amplified and easily measured. Flow-cytometric analysis revealed that PSA levels on Chinese hamster ovary (CHO) cells decrease with increasing pCO(2) in a dose-dependent manner, independent of any change in NCAM content. The results are highly pH-dependent, with a greater decrease in PSA at higher pH. By manipulating medium pH and pCO(2), we showed that decreases in PSA correlate well with bicarbonate concentration ([HCO(3)(-)]). In fact, it was possible to offset a 60% decrease in PSA content at 120 mm Hg pCO(2) by decreasing the pH from 7.3 to 6.9, such that [HCO(3)(-)] was lowered to that of control (38 mm Hg pCO(2)). When the increase in osmolality associated with elevated [HCO(3)(-)] was offset by decreasing the basal medium [NaCl], elevated [HCO(3)(-)] still caused a decrease in PSA, although less extensive than without osmolality control. By increasing [NaCl], we show that hyperosmolality alone decreases PSA content, but to a lesser extent than for the same osmolality increase due to elevated [NaHCO(3)]. In conclusion, we demonstrate the importance of pH and pCO(2) interactions, and show that [HCO(3)(-)] and osmolality can account for the observed changes in PSA content over a wide range of pH and pCO(2) values.

Role of nucleotide sugar pools in the inhibition of NCAM polysialylation by ammonia

Ammonia in animal cell cultures has been shown to specifically inhibit terminal sialylation of N‐ and O‐linked oligosaccharides of glycoproteins. For example, we have previously shown that as little as 2.5 mM NH4Cl can decrease neural cell adhesion molecule (NCAM) polysialylation in both small cell lung cancer (SCLC) and Chinese hamster ovary (CHO) cells. Besides its potential involvement in SCLC metastasis, polysialic acid (PolySia) is a sensitive marker for measuring changes in sialylation. The role of UDP‐N‐acetylglucosamine (UDP‐GlcNAc) in ammonias inhibition of NCAM polysialylation was examined by adding glucosamine (GlcN) and uridine (Urd) to the cultures. This bypassed feedback inhibition of GlcN‐6‐P synthase and increased UDP‐GlcNAc content by 25‐fold in SCLC cells. After 3 days, PolySia levels were reduced to 10% of control with little effect on NCAM protein content. The extensive decrease in PolySia was confirmed in CHO cells. The effects of GlcN or Urd alone were less extensive, lending support to a specific role for UDP‐GlcNAc in inhibition by ammonia. By comparison, 20 mM NH4Cl decreased PolySia content by 45% and increased UDP‐GlcNAc in SCLC cells by 2‐fold. The discrepancy between the {GlcN+Urd} and NH4Cl effects on UDP‐GlcNAc and PolySia suggests that accumulation of UDP‐GlcNAc is only partially responsible for decreased polysialylation in response to NH4Cl. In an attempt to increase NCAM polysialylation, N‐acetylmannosamine and cytidine were added to cultures in order to circumvent the feedback inhibition of CMP‐sialic acid synthesis. However, this only slightly increased PolySia levels and failed to counter ammonias inhibition of NCAM polysialylation.

AMMONIA INHIBITS NEURAL CELL ADHESION MOLECULE POLYSIALYLATION IN CHINESE HAMSTER OVARY AND SMALL CELL LUNG CANCER CELLS

Ammonia is a major concern in biotechnology because it often limits recombinant protein production by animal cells. Conditions, such as ammonia accumulation, in large‐scale production systems can parallel those that develop within fast‐growing solid tumors such as small cell lung cancer (SCLC). Ammonias specific inhibition of the sialylation of secreted glycoproteins is well documented, but it is not known how ammonia affects membrane‐bound proteins, nor what role it may have on important glycosylation determinants in cancer. We therefore examined the effects of NH4Cl on polysialic acid (PolySia) in the neural cell adhesion molecule (NCAM). By using flow cytometry combined with two NCAM antibodies, one specific for the peptide backbone and another that recognizes PolySia chains, we show that ammonia causes rapid, dose‐dependent, and reversible inhibition of NCAM polysialylation in Chinese hamster ovary (CHO) and SCLC NCI‐N417 cells. The decrease in PolySia was accompanied by a small increase in NCAM, suggesting that the changes were specific to the oligosaccharide. Inhibition by ammonia was greater for CHO cells, with PolySia cell surface content decreasing to 10% of control after a 4‐day culture with 10 mM NH4Cl, while N417 cell PolySia was reduced by only 35%. Ammonia caused a 60% decrease in the CHO cell yield from glucose, while N417 cells were barely affected, suggesting that increased resistance to ammonia by N417 cells is a global rather than glycosylation‐specific phenomenon. The data presented show that the tumor microenvironment may be an important factor in the regulation of PolySia expression. J. Cell. Physiol. 177:248–263, 1998.

Quantitative 19F NMR study of trifluorothymidine metabolism in rat brain.

Metabolism of trifluorothymidine (TFT) and its transport across the blood–brain barrier (BBB) has been measured quantitatively in rats by fluorine‐19 nuclear magnetic resonance spectroscopy (19F NMR). It is demonstrated that TFT crosses the BBB in micromolar quantities and is metabolized in brain tissue primarily to its free base trifluoromethyluracil (TFMU) by the enzyme thymidine phosphorylase (TP). It is further proposed that the rate of TFMU production can be used as a measure of cerebral TP. The glycols of both TFMU, and to a lesser degree TFT, are generated via an oxidative route. In contrast, the major pathway for hepatic metabolism of this compound is through reduction of the nitrogen base moiety and generation of 5‐6‐dihydro species followed by ring degradation. Thus, in addition to TFMU as well as the dihydroxy (glycol)‐, and the dihydro‐species of both TFT and TFMU, α‐trifluoromethyl‐β‐ureidopropionic acid (F3MUPA) and α‐trifluoromethyl‐β‐alanine (F3MBA) were detected in liver extracts. The total metabolite levels in liver were 2–5 times higher than in the brain. Low levels of fluoride ion were detected in all the extracts from brain and liver, as well as blood and urine. This study characterizes TFT as a potential chemotherapeutic agent for use against brain tumors. Copyright

Phase I trial of fixed dose rate infusion gemcitabine with gefitinib in patients with pancreatic carcinoma.

Purpose: To determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of gemcitabine using a fixed dose rate infusion (FDRI) in combination with gefitinib in patients (pts) with pancreatic adenocarcinoma (PCa). Patients and methods: Patients with advanced PCa were given gemcitabine at the FDRI of 10 mg/m2/min IV on Days 1, 8, and 15 of a 28-day cycle. Dose levels of 1000, 1200, and 1500 mg/m2 were evaluated. Oral gefitinib 250 mg was given daily. DLTs were defined as 2 instances of Grade 3 hematologic or 4 nonhematologic or any Grade 4 hematologic toxicity. At least 4 patients were treated at each dose level. Dose escalation occurred in the absence of DLTs. Results: Five women and 8 men were enrolled. Median age was 59 and performance status 1. All had metastatic disease. Four patients received prior adjuvant chemoradiation for PCa, and one chemotherapy for lung cancer. Median cycles were 4 per patient. The MTD was 1,200 mg/m2. Toxicity was predominantly hematologic. At 1,500 mg/m2, 1 patient had Grade 4 granulocytopenia and 3 patients Grade 3 granulocytopenia. Overall, 8 patients (60 percent) developed Grade 1 or 2 acneiform rashes. One patient had Grade 3 vomiting; no significant diarrhea or liver toxicity was seen. There were no objective responses seen. Median time to progression and overall survival were 4.57 months and 7.13 months, respectively. Conclusion: Combining FDRI gemcitabine with gefitinib is feasible and tolerable. The recommended dose of gemcitabine is 1,200 mg/m2 when used with gefitinib 250 mg daily.

High-dose aminothiadiazole in advanced colorectal cancer - An Illinois Cancer Center phase II trial

SummaryThirty-three patients with advanced colorectal carcinoma were entered on a phase II trial of weekly IV aminothiadiazole (175 mg/m2 escalated to 200 mg/m2) with concomitant allopurinol and non-steroidal anti-inflammatory agents (NSAIDs). Toxicity was predominantly GI, cutaneous, and chest pain/dyspnea. Twenty-five percent of patients had grade 3 or 4 toxicity. There were no responses in 27 evaluable patients. Median survival was 12 months. Aminothiadiazole, at higher doses than used in previous reports, when given with NSAIDs, had no significant activity against large bowel cancer.