Medial vs lateral wall pyriform sinus carcinoma: implications for management of regional lymphatics.

BACKGROUND: Our purpose in performing this review is to analyze the relationship between the anatomic site of hypopharyngeal lesions and the pattern of recurrent disease, particularly cervical recurrence. This clinical information can subsequently be used to influence treatment options. METHODS: The records of 169 patients with carcinoma of the hypopharynx treated between 1975 and 1986 were reviewed retrospectively. Patients were classified as having medial wall pyriform sinus disease (MP) lesions, lateral wall pyriform sinus (LP) lesions, posterior wall (PW), or postcricoid (PC) lesions. All patients were followed a minimum of 36 months. Patients with evidence of recurrent carcinoma were characterized according to the site of recurrence. RESULTS: Recurrent carcinoma in the hypopharynx was noted in seven (4%) of 169 patients. Cervical metastases was the sole site of failure in 27 (16%) of 69 patients, whereas distant metastases developed in 25 (15%) of 169 patients. Failure in the contralateral unoperated neck occurred in 14% (10/71) of patients with MP lesions and, in contrast, 5% (4/76) LP patients. This difference was statistically significant (p = 0.04). Radiotherapy was not effective in preventing cervical recurrence in 20 (74%) of 27 overall neck failures and 11 (79%) of 14 contralateral NO neck failures. CONCLUSION: These data suggest that patients with carcinoma involving the MP are at greater risk for contralateral cervical metastases. We recommend bilateral neck dissection be offered to patients with MP lesions.

Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase.

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32P-ACC phosphorylated by the casein kinases was identified.

Phosphorylation of acetyl-coenzyme A carboxylase by casein kinase I and casein kinase II.

Two cAMP-independent acetyl-CoA carboxylase (ACC) protein kinases have been partially purified from rat liver cytosol and microsomal extracts. The first kinase, present in greatest activity in microsomal extracts, appears to be identical to casein kinase I by characteristic molecular size on gel filtration (Mr 40,000) and sodium dodecyl sulfate-gel electrophoresis (Mr 34,000), autophosphorylation of this single subunit, inability to efficiently utilize GTP, and resistance to inhibition by heparin and 2,3-diphosphoglycerate. The second kinase, predominant in cytosol, appears to be identical to casein kinase II by characteristic molecular size on gel filtration (Mr 150,000), an autophosphorylated subunit of Mr 25,000, a Km for GTP nearly equal to that of ATP, inhibition by heparin and 2,3 DPG, and relative substrate specificity. Despite the incorporation of up to 2 mol 32P/mol carboxylase subunit (kinase I) and 0.6 mol/subunit (kinase II), phosphorylation by either kinase causes no change in carboxylase activity. The site(s) phosphorylated by each kinase and by the cAMP-dependent protein kinase on carboxylase appear to be clustered on a Mr 16,000 cyanogen bromide peptide that is readily released on incubation with trypsin. The potential roles of these kinases in the regulation of ACC remain to be clarified.

Stimulation of site-specific phosphorylation of acetyl coenzyme A carboxylase by insulin and epinephrine.

32P-labeled acetyl-CoA carboxylase was isolated from 32P-labeled rat epididymal fat pads by avidin-Sepharose affinity chromatography after exposure to epinephrine and insulin. Epinephrine led to an inactivation of the isolated enzyme by a reduction of Vmax, while the insulin stimulation observed in crude extracts did not survive enzyme purification. Both insulin and epinephrine caused only small increases in total 32P content of the enzyme. However, mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography revealed that epinephrine and insulin stimulated the phosphorylation of 32P-peptides specific for each hormone. The major 32P-peptide phosphorylated by epinephrine co-migrated with the major 32P-peptide phosphorylated in vitro by the cAMP-dependent protein kinase, while the 32P-peptide phosphorylated in response to insulin co-migrated with that phosphorylated by casein kinase-I and casein kinase-II. The effects of epinephrine on carboxylase activity and phosphorylation can thus be accounted for by the expected epinephrine-induced activation of the cAMP-dependent protein kinase. While the increase in site-specific phosphorylation caused by insulin cannot be directly linked to insulin-induced activation in crude extracts, these data suggest that casein kinase-I and/or casein kinase-II may mediate the insulin-stimulated phosphorylation of acetyl-CoA carboxylase.

Regulation of acetyl-CoA carboxylase by guanine nucleotides.

Acetyl-CoA carboxylase, a major rate-limiting enzyme for fatty acid synthesis, is subject to acute regulation by both allosteric modulation and covalent enzyme phosphorylation. Because citrate activation of the enzyme in vitro requires citrate concentrations far in excess of intracellular levels, we have attempted to identify other ligands which might mediate carboxylase activity. Heated liver extracts contain a potent endogenous activator of carboxylase assayed in dialyzed high speed liver supernatant; the activator elutes behind the salt volume of a Bio-Gel P-6 gel filtration column, is destroyed by alkaline phosphatase, and is adsorbed by charcoal. This activator activity is shared by several guanine nucleotides (5'-GTP, 5'-GDP, 5'-GMP, and 3':5'-cyclic GMP). Further separation of the endogenous activator by high pressure liquid chromatography reveals a carboxylase-activating compound which co-elutes with 5'-GMP. The guanine nucleotides are potent activators of carboxylase activity at intracellular nucleotide concentrations and permit expression of maximal enzyme velocity at cytosolic citrate concentrations. However, we have been unable to document any effects of guanine nucleotides on isolated rat liver acetyl-CoA carboxylase. While the mechanisms of these effects remain to be elucidated, they suggest that the guanine nucleotides may be important intracellular regulators of carboxylase activity and of fatty acid synthesis.

Microsomal acetyl-CoA carboxylase: evidence for association of enzyme polymer with liver microsomes.

Fatty acid synthesis is traditionally viewed as being confined to the cytosolic cellular fraction, although a substantial body of data indicates that both microsomes and mitochondria are capable of initiating fatty acid synthesis and may contain acetyl-CoA carboxylase [acetyl-CoA:carbon-doxide ligase (ADP-forming), EC 6.4.1.2], fatty acid synthetase, and ATP-citrate lyase [ATP citrate (pro-3S)-lyase; ATP:citrate oxaloacetate-lyase (pro-3S-CH2COO- leads to acetyl-CoA; ATP-dephosphorylating), EC 4.1.3.8] activities. We have identified 32P-labeled acetyl-CoA carboxylase and 32P-labeled ATP-citrate lyase by immunoprecipitation of a rat hepatocyte microsomal preparation. In the transition between the fasting state (low rates of lipogenesis) and fasting/re-feeding (high rates), the fraction of total cytosolic plus microsomal acetyl-CoA carboxylase in the microsomes increases from 6% to 43%, whereas the microsomal proportion of total fatty acid synthetase and ATP-citrate lyase remains approximately 10%. Microsome isolation conditions favoring carboxylase polymerization (presence of citrate) promote microsomal association, whereas conditions favoring enzyme protomerization (malonyl-CoA, preincubation with cyclic AMP/ATP/Mg2+) diminish this association. The microsomal enzyme has a 5-fold higher specific activity than the cytosolic enzyme as determined by immunotitration. Sucrose density gradient analysis of the microsomal fraction indicates that a substantial portion of carboxylase activity sediments with marker enzymes for endoplasmic reticulum, plasma membrane, Golgi apparatus, and outer mitochondrial membrane, while cytosolic enzyme or isolated enzyme incubated under polymerizing conditions does not penetrate the gradient. These data suggest that the microsomes may be a significant locus of fatty acid synthesis initiated with association of acetyl-CoA carboxylase polymer with this fraction.