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High expression of thymidylate synthase (TS) is allegedly associated with the chemoresistance to 5-fluorouracil (5-FU) in colorectal cancers. However, low TS expression does not necessarily imply chemosensitivity. Inactivation of p16(INK4a) correlates with poor prognosis in various cancers. We immunohistochemically evaluated the relationship between the expression of TS, p16(INK4a), CDK4 and cyclin D1 and the effect of 5-FU-based chemotherapy in colorectal cancers. After antigen retrieval, immunoperoxidase staining was performed on the paraffin-embedded, biopsy and surgical specimens of 37 advanced colorectal cancers preoperatively treated with peroral administration of 5-FU derivatives. As a control group, 31 colorectal cancers without preoperative treatment were analyzed. High TS expression was found in 23 (74%) of 31 tumors resected from histological non-responders and in 19 (61%) of 31 controls but in none of six responders. High p16(INK4a) expression was seen in 83% of the responders, 52% of the non-responders and 32% of the controls. The TS-low/p16(INK4a)-high phenotype was noted in 83% of the responders, but only in 3% of the non-responders (P = 0.0001). Induction of p16(INK4a) expression after chemotherapy was predominantly seen in the responders. Neither CDK4 nor cyclin D1 expression was related to the chemotherapeutic effects. In conclusion, the combination of low expression of TS and induction of p16(INK4a) after chemotherapy can be important indicators of the sensitivity to 5-FU-based chemotherapy in colorectal cancers.

Phosphorylated sites of calf thymus H2B histone were investigated with a catalytic fragment of 72 kDa protein-tyrosine kinase (p72syk). Three of five tyrosine residues in H2B histone can be phosphorylated by this kinase. In this analysis, H2B histone was thoroughly phosphorylated in vitro with [gamma-32P]ATP and the kinase, and then digested with a lysylendopeptidase. The resulting radioactive phosphopeptides were separated by a reverse-phase column on high performance liquid chromatography. Subsequent sequential Edman degradation of the purified phosphopeptides revealed that 40Y, 83Y and 121Y were phosphorylated. 121Y is the major phosphorylated residue in H2B histone. No phosphorylation was detected in 37Y and 42Y. Although the consensus sequence was not defined from these analyses, our data suggest that higher-order structure(s) in addition to primary one may participate in recognition of H2B histone by this protein kinase.

1. Endogenous phosphate acceptor proteins by cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) were studied using subcellular fractions of porcine spleen and supernatant fraction of rat various tissues. 2. At least 13 phosphate acceptor proteins ranging from 94 to 26 kDa were observed in all but mitochondrial subcellular fractions. 3. Among the supernatant fraction of rat tissues, brain, testis and spleen contained many phosphate acceptor proteins. 4. The most heavily phosphorylated band of around 55 kDa which was commonly recognized among various tissues was confirmed as tubulin by the immunoreactivity with anti-tubulin antibody. 5. The results obtained in this paper indicate that CPTK-40 has the potential to catalyze the phosphorylation of numerous endogenous proteins including tubulin.

Effect of membrane phospholipids on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) has been studied. Using poly(Glu Na, Tyr)4:1 as a substrate, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine had stimulatory effects on that phosphorylation activity, however phosphatidic acid had inhibitory and phosphatidylinositol had no effects. Similar results were obtained using[Val5]angiotensin II as a substrate. On the other hand using basic protein (H2B histone and myelin basic protein) as substrates, phosphatidic acid stimulated the activity of CPTK-40, while phosphatidylinositol inhibited the activity. Phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine caused different effect on the activity of CPTK-40 depending on the substrate employed. However using acidic protein (tubulin and casein) as substrates, the activity of CPTK-40 was neither stimulated nor inhibited by any phospholipids. These results suggest that phospholipids may modulate the activity of CPTK-40.