David A. Potter

From July 1975 to December 1982, 563 patients were referred to the Surgery Branch of the National Cancer Institute with the diagnosis of soft-tissue sarcoma. Three hundred and seven of these patients had fully resectable, localized high-grade soft-tissue sarcomas and were treated at the National Cancer Institute using standard protocols with surgery alone, or in combination with chemotherapy and/or radiotherapy. An aggressive surgical approach was undertaken in the management of patients who subsequently developed recurrent disease. These 307 cases have been reviewed, with a median duration of follow-up of 30 months, to determine the frequency of recurrent disease, the patterns of recurrence, and the impact of surgery on the survival of patients who developed recurrent disease. Disease recurred in one hundred seven patients (107/307, 35%), with a median disease-free interval of 18 months (range, 0.5 to 72.0 months). The frequency of recurrence by site of primary sarcoma was extremity, 31% (65/211); head and neck, 33% (4/12); trunk, 40% (17/42); retroperitoneum, 47% (17/36); and breast, 67% (4/6). Isolated pulmonary metastatic disease was the most common pattern of initial recurrence (56/107, 52%) followed by isolated local recurrence (21/107, 20%). Single other sites of recurrence and multiple concurrent sites of recurrence each accounted for 14% (15/107) of all initial recurrences. The relative frequency of each of these four patterns of recurrence varied with the site of the primary sarcoma. The outcome for patients with recurrent disease depended on the site of recurrence, rather than on the site of the primary sarcoma. Sixty-six patients (66/107, 62%) with recurrent disease were rendered surgically disease-free with the first recurrence, including 40 (40/56, 72%) patients with isolated pulmonary metastases, 20 patients (20/21, 96%) with isolated local recurrences, five patients (5/15, 33%), with isolated other sites of recurrence and one patient (1/15, 7%) with multiple sites of initial recurrence. Following surgical resection, the actuarial three-year survival for the 66 patients rendered disease-free was 51%. The median survival for the 41 patients not rendered surgically disease-free with the first recurrence was only 7.4 months. Thirty of the sixty-six patients (30/66, 45%) rendered disease-free with the first recurrence remained disease-free at follow-up, with a median follow-up of 28 months from the time of resection of the first recurrence. The remaining 36 patients (36/66, 55%) subsequently recurred, with a median disease-free interval of 7.3 months.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.

How m-calpain is activated in cells has challenged investigators because in vitro activation requires near-millimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.

Serum amyloid A (SAA) is an apolipoprotein produced by the liver in response to inflammation; the levels of SAA mRNA and SAA protein increase at least 500-fold within 24 h. We have obtained clones of all three genes and pseudogene that make up the murine SAA gene family. Two of the genes have 96% sequence homology over their entire length, including introns and flanking sequences 288 base pairs (bp) 5' and 443 bp 3' to the genes: an overall length of 3215 bp. The sharp boundaries between homologous and nonhomologous sequences and the absence of interspersed repeated sequences there suggest that conversion has occurred between these two genes. The homologous regions are bounded by short inverted repeats containing alternating purine and pyrimidine residues, as described for other gene conversion units. The third SAA gene has evolved separately, although all are closely linked on chromosome 7. Comparison of the upstream regions of the SAA genes with those of the rat fibrinogen genes, whose expression is also induced by inflammation, reveals sequences common to all six genes which are very improbable on a random basis.

Major changes in the mRNA population of murine liver occur after administration of bacterial lipopolysaccharide, an agent that causes increases in the concentrations of acute-phase serum proteins. The mRNA for one of these, serum amyloid A, is increased at least 500-fold compared to the normal level. It becomes one of the most abundant hepatic mRNAs, and serum amyloid A synthesis comprises about 2.5% of total hepatic protein synthesis in the acute-phase response. Its synthesis is tissue-specific in that amyloid A mRNA was not detected in the kidney, an important site of amyloid fibril accumulation. The protein synthesized in largest amount by acute-phase liver tissue in culture is cytoplasmic actin. Its relative rate of synthesis is increased about 5-fold compared to the normal tissue; that of serum albumin is decreased to about one-third of its normal rate. The concentration of mRNA for serum albumin is decreased by a similar amount. Starting with induced liver RNA, we have constructed a recombinant plasmid containing most of the DNA sequence encoding the serum amyloid A polypeptide.