Salivary-type hyperamylasemia in primary lung cancer: Observation of a possible precursor of the salivary-type isoamylase

Although recent studies by column chromatography and electrophoresis showed that elevated amylase activity in the body fluids and tumor extracts in patients with primary lung cancer was mainly in the salivary-type isoamylase, neither mechanism nor nature of elevation of amylase have been clarified yet. This study was undertaken in order to make clear the nature of amylase elevated in the body fluids and tumor extracts of patients with primary lung cancer. In addition, amylase contained in the extracts of normal lungs and diseased lungs of patients who had died of various diseases was also studied. Our results revealed that amylase elevated in the body fluids and tumor extracts in patients with primary lung cancer was the salivary-type isoamylase and, moreover, that small amounts of salivary-type isoamylase in normal lung tissues and large amounts in diseased lung tissues were also contained. These facts suggested that salivary-type isoamylase was physiologically contained in normal lung tissues and might be activated through pathological processes, such as inflammation, circulatory disturbance or tumor formation. Two peculiar isoamylases with cathodic mobility on polyacrylamide gel electrophoresis were found. One of them was so unstable that it was converted to the stable salivary-type isoamylase, suggesting the precursor of human salivary isoamylase.     

Simultaneous production of amylase and adrenocorticotropin by lung cancer--with special reference to electron microscopic and immunohistochemical studies

The tumor tissue from a lung cancer patient who showed elevated serum amylase and adrenocorticotropin was investigated ultrastructurally and immunohistochemically. On microscopic examination, the tumor was diagnosed as small-cell carcinoma. Electron microscopy revealed that some of the tumor cells possessed small endocrine-like dense cored granules. The tumor cells also contained large zymogen-like granules within the cytoplasm, which possessed microvilli and formed the lumen, indicating their adenocarcinomatous differentiation. An electrophoretic analysis of the serum amylase showed that the major amylase elevated was of the salivary type. Immunohistochemical staining by the antihuman salivary amylase antibody disclosed that various portions of the tumor actually contained the salivary amylase. The evidence suggests that the small-cell carcinoma cells showed "confused differentiation", thereby expressing amylase and ACTH simultaneously.     

Amylase: a disease activity index in multiple myeloma?

This study reports a case of a patient with lambda-light chain multiple myeloma who developed a high hyperamylasaemia of the salivary type during the disease and soon afterwards died. Ectopic production of amylase by myeloma cells has been described in a few cases and demonstrated by tissue culture and immunohistochemical techniques. The common characteristics of these cases were: salivary amylase isoenzyme increase, high tumor mass, extensive extra-medullary spread, extensive bone destruction and poor prognosis. In patients with amylase-producing multiple myeloma, the onset of hyperamylasaemia heralds a rapid disease progression; therefore, in these patients, a simple test such as serum amylase may represent a reliable disease activity index and provide an additional prognostic information.     

A case of amylase-producing ovarian tumor

A 51-year-old woman with ovarian carcinoma with hyperamylasemia is reported. She was admitted to Osaka General (JNR) Hospital in July 1982. A cystic tumor was palpated in the pelvis. Serum amylase, mainly the salivary type, in electrophoresis, had increased to 1583 Smith-Roe units/dL, while the pancreas was normal on ultrasonography and CT. Chemotherapy (MFC) was performed, and laparotomy revealed bilateral ovarian tumors. Histologically, they were serous cystadenocarcinoma. Serum and urinary amylase values dropped to the normal range after chemotherapy and surgery, and varied directly with the clinical course. Immunohistochemically, amylase was demonstrated in the tumor tissue by the PAP method. In conclusion, amylase was a useful marker in the diagnosis and follow-up of the present patient.     

Biological and biochemical properties of Nonidet P40-solubilized and partially purified tumor-specific antigens of the transplantation type from plasma membranes of a methylcholanthrene-induced sarcoma.

Tumor-specific transplantation antigen (TSTA) was solubilized from cell membranes of sarcoma Meth-A with non-ionic detergent Nonidet P40. Soluble TSTA was partially characterized by chromatographic separation and electrophoresis. The antigen responsible for tumor rejection activity had a molecular weight of approximately 70,000 daltons in the presence of detergent and an electrophoretic mobility of alpha-globulin. TSTA was well separated from mouse histocompatibility antigen H-2 by a sequence of procedures, including gel filtration, lectin affinity chromatography, column electrophoresis, and rechromatography on agarose, showed only three major bands on polyacrylamide gel electrophoresis. TSTA was specific for sarcoma Meth-A.     

Small cell undifferentiated carcinoma of the minor salivary gland containing exocrine, neuroendocrine, and squamous cells

The light microscopic, electron microscopic and immunohistochemical features of a small cell undifferentiated carcinoma of the minor salivary gland are presented. The tumor was composed predominantly of undifferentiated small cells with focally admixed neuroendocrine, exocrine and squamous cells, occasionally arranged in an organoid manner. The presence of vasoactive intestinal polypeptide in the tumor was found immunohistochemically. In addition, the tumor cells stained with Grimelius' impregnation. Immunohistochemically the tumor contained cells that reacted positively with the antibodies to carcinoembryonic antigen, 66K keratin polypeptide, or human salivary amylase. These findings indicate that a small cell undifferentiated carcinoma of the minor salivary gland, reported here, exhibits focally multidirectional differentiation as well as neuroendocrine cell derivation.     

Elevated activity of beta-hexosaminidase and sulfhydryl modification in the B-variant of human lung cancer

Activities of beta-hexosaminidase A and beta-hexosaminidase B (Hex B) were measured both in human lung carcinoma and the adjacent normal tissues of 47 patients. The specific activity of total beta-hexosaminidase in the tumors was considerably higher than in the adjacent normal tissues, irrespective of histological types. In isoelectric focusing experiments, Hex B purified from normal lung exhibited a single peak with an isoelectric point (pI) of 7.9, while Hex B purified from adenocarcinoma contained two forms with pI 7.6 and 7.9. With respect to heat stability, Hex B from the normal lung was very stable at 52 degrees, while the tumor Hex B (mixture of pI 7.6 and 7.9 forms) was unstable. After treatment of the tumor enzyme with dithiothreitol, heat stability was restored. When the tumor pI 7.6 form was treated with dithiothreitol and subjected to polyacrylamide gel electrophoresis, the enzyme converted to a pI 7.9 form similar to that of the normal lung. Determination of the sulfhydryl group of the tumor pI 7.6 form under nondenaturing conditions showed that the enzyme had some easily reducible disulfide bonds on its surface. These findings indicate that the formation of mixed disulfide bonds in the tumor Hex B increases the net negative charge and results in the appearance of a heat-labile form.     

The expression of placental glutathione-s-transferase (gst-pi) in human normal salivary-glands and tumors

Immunohistochemical localization of placental glutathione S-transferase (GST-pi) in normal and inflammatory salivary glands (74 cases) and tumors (71 cases) was studied. In the normal and inflamed salivary glands of all locations (including major and minor salivary glands), the ductal epithelial cells showed moderate to strong GST-pi staining, myoepithelial cells showed weak staining and the acinar cells were negative. The staining pattern of the tumor cells was similar to that of their normal counterparts. The tumor cells were generally positive with GST-pi staining except those tumor cells demonstrating acini differentiation (serous cells in acinic cell carcinoma, mucous cells in mucinous cystadenoma and mucoepidermoid carcinomas). Most of the salivary gland tumors, benign or malignant, showed a weak GST-pi staining. Only mucoepidermoid carcinomas demonstrated significantly increased GST-pi reactivity compared to other tumors (p<0.0001), which may be a reflection of both malignancy and squamous differentiation of the tumor. The marked increase in GST-pi activity in mucoepidermoid carcinomas may be useful in serological screening of recurrent and metastatic mucoepidermoid carcinomas.     

Collagen cell attachment protein from rat hepatoma cells

This paper describes the isolation and partial characterization of a collagen cell attachment protein from the spent culture medium of rat hepatoma cells. When compared with serum fibronectin, this attachment protein differed in several biochemical parameters. The hepatoma attachment protein was partially purified by adsorbing and eluting from an inorganic gel, magnesium oxide. Cell adhesive activity may routinely recovered at levels of 10 to 30%, and a 2000-fold purification was attained. The hepatoma attachment protein was shown to be sensitive to trypsin and chymotrypsin, to be heat inactivated at 61 degrees, to have a molecular weight of 58,000, to have an isoelectric point of 4.1, to show an electrophoretic mobility on cellulose acetate of approximately one-half that of fibronectin, and not to cross-react with antifibronectin antisera.     

An autopsied case of an amylase-producing lung cancer

An autopsy involving a case of a amylase-producing lung cancer is reported. The patient was a 80-year-old female, who had been admitted with dyspnea and right pleural effusion. Cytological examination of this pleural effusion revealed adenocarcinoma cells. On clinical examination, owing to a lack of particular changes in the other organs, lung cancer was suspected. She received chemotherapy but died of respiratory failure. Serum examination showed a high amylase level throughout her clinical course. The autopsy revealed a diffuse gray-whitish tumor in the right lower lobe with metastasis to the lungs, diaphragm, epicardium, spleen, liver, colon, and the regional lymph-nodes. Histologically, a well-differentiated papillary adenocarcinoma was revealed in the primary and metastatic areas. Immunohistochemical stains showed positive salivary gland-type amylase activity in the cytoplasms of the tumor cell.     

Serum amylase is a sensitive tumor marker for amylase-producing small cell lung cancer?

A 68-year-old male smoker was diagnosed as having amylase-producing small-cell lung cancer (SCLC). The serum amylase level was elevated, at 1756 IU/l, and the isozyme pattern was salivary type. Serum levels of “the tumor markers” CEA and NSE were 10.0 ng/ml and 22.6 ng/ml, respectively, but the level of pro-GRP was within the normal range. He was treated with combination chemotherapy of carboplatin and irinotecan. After completion of the chemotherapy, the serum amylase level decreased below the cutoff range and a computed tomography (CT) scan of the chest revealed marked reductions of the tumor in the primary site and in the lymph node metastasis. In November 2003, he was noted to have a slightly raised amylase level, of 168 IU/l, and raised levels of tumor markers. At this time, a CT scan, bone scintigraphy, and magnetic resonance imaging (MRI) of the brain demonstrated no recurrence. However, in December, MRI of the brain showed multiple metastases, and the recurrence of SCLC was thus confirmed. For the treatment of disease progression, the same regimen of chemotherapy as that given initially was administered. CT imaging revealed a partial response in the primary site and lymph node metastasis, and the serum amylase level decreased to 91 IU/l. After the completion of the second chemotherapy regimen, he underwent cranial irradiation and further chemotherapy. However, unfortunately, he died owing to deterioration of lung cancer. In this patient, the serum amylase level was found to be a highly sensitive marker of lung cancer.     

Arginine vasopressin promoter regulation is mediated by a neuron-restrictive silencer element in small cell lung cancer.

Arginine vasopressin (AVP) is often expressed in small cell lung cancer (SCLC), and a 65-bp AVP minimal promoter fragment is sufficient to restrict activity to SCLC in vitro. We now describe a motif with homology to the neuron-restrictive silencer element (NRSE) within this fragment. Electrophoretic mobility shift analysis demonstrated that multiple specific complexes are bound by this motif. These complexes are cross-competed with a characterized SCG10 NRSE probe and do not bind to the AVP probe with a specific mutation in the NRSE. The complexes vary in mobility between lung tumor cell lines, showing different levels of AVP expression, and some are differentially bound in SCLC. Overexpression of a neuron-restrictive silencer factor expression construct can silence reporter gene expression supported by the AVP promoter in SCLC, although this was dependent on both the level of endogenous AVP expression in the cells and putative enhancer elements in larger promoter constructs. Activation of the proximal AVP promoter in SCLC is therefore proposed to, at least partially, rely on modulation of normal repressor activity at the NRSE.     

An amylase-producing serous cystadenocarcinoma of the ovary

A patient with an amylase-producing serous cystadenocarcinoma of the ovary had elevated serum and urine amylase levels and high levels of amylase in pleural and ascitic fluids. Serum and urine amylase levels reflected both surgical removal of tumor mass and response to chemotherapy. Tumor homogenates had pronounced amylase activity. Salivary type amylase isozyme patterns were found in electrophoresis of samples from all sources. Ascites tumor cells were successfully cultured and salivary type amylase was found in the culture media throughout 5 passages. The tumor was classified by light microscopy as poorly differentiated serous cystadenocarcinoma. Ultrastructural studies on the tumor were consistent with that diagnosis. Amylase was detected in the cells of the tumor examined by the immunoperoxidase technique.     

Amylase-producing ovarian cancer treated with postoperative QF chemotherapy--a case report

A case of an amylase-producing ovarian cancer in 69-year-old woman has been investigated by light and electron microscopy, as well as by amylase isozyme analysis. Serum and urinary amylase levels were found to be elevated, especially by an amylase isozyme analysis of the tumor homogenate which revealed a salivary type of cancer. The serum amylase level and its isozyme pattern returned to normal following therapy. Pathological examination revealed a serous cystadenocarcinoma. The ultrastructure of this tumor resembled that of other ovarian serous cystadenocarcinomas. No zymogen granules were recognized. Thus, a biochemical analysis of amylase isozyme was found to be a useful tumor marker for diagnosis and treatment of an amylase-producing ovarian serous neoplasm.     

Heterogeneity of sialoglycoproteins purified from normal and malignant cells

We have identified previously a group of sialoglycoproteins with an apparent molecular weight of 110,000, which appear homogeneous by sodium dodecyl sulfate:polyacrylamide gel electrophoresis but exhibit isoelectric point heterogeneity by two-dimensional polyacrylamide gel electrophoresis. This technique demonstrated that there are multiple glycoproteins of similar molecular weight which differ between normal cells and transformants. We have now purified glycoproteins by concanavalin A:Sepharose chromatography and preparative polyacrylamide gel electrophoresis from both a nontumorigenic normal mouse fibroblast line, A31, and a highly malignant nonimmunogenic transformant, 3T12T. Differences in the isoelectric point distribution of the sialoglycoproteins which were observed between the normal and transformed two-dimensional gel patterns using crude membranes could also be demonstrated with the purified glycoproteins. Treatment of the isolated sialoglycoproteins with neuraminidase to remove sialic acid resulted in significant isoelectric point shifts but did not eliminate all of the heterogeneity. Even following neuraminidase treatment, the purified glycoprotein fraction upon isoelectric focusing showed differences in patterns between normal and transformed cells. Preliminary characterization of the alterations seen in the two cell lines are presented and show decreased fucose and increased sialic acid in transformed cell glycoprotein.     

Cancer-specific proteins and their application in the diagnosis of lung cancer

Thin slab PAGE was used to analyze 588 normal serum samples, 134 lung cancer samples and 68 noncancer lung diseases samples to determine whether distinct protein bands could be classified as tumor markers. Three distinct bands were noted. The band most frequently seen in the sera of lung cancer patients was of the molecular weight of 175, 000 Doltons while a band of 115,000 Doltons appeared with the sera of normal and noncancerous lung disease patients. These bands were termed “tumor positive” and “tumor negative” markers respectively. The tumor negative marker protein reacted readily with anti-C3, and it was called complement-related protein or Rc3. The tumor positive marker corresponded to lung cancer in 78.4% of cases; the false positive rates for normal and non-cancer lung diseases were 6% and 11%, respectively. The sensitivity of this method was 78.4% (S=78%); specificity was 86% (F=86%.)     

Purification and partial amino acid sequence of rabbit tumor necrosis factor

Good production of tumor necrosis factor (TNF) in the rabbit was obtained using Propionibacterium acnes IID 912 as a priming agent and subsequent administration of lipopolysaccharide. The physicochemical characteristics of rabbit TNF were very similar to those of murine TNF. The molecular weight of rabbit TNF was 39,000 as estimated by gel filtration, and 18,000 by SDS-PAGE. The isoelectric point was determined as pH 4.0 by isoelectric focusing. Rabbit TNF was stable within the pH range of 5.5 to 11.0, and was stable at 56 degrees C for 8 hr. It was digested by trypsin, pancreatic protease and elastase, but was resistant to neuraminidase. The amino acid sequence of rabbit TNF was determined as Ser-Ala-Ser-Arg-Ala-Leu- .... Monoclonal antibody against rabbit TNF completely inhibited both the in vivo and in vitro activity of rabbit TNF. However, this antibody could not inhibit the action of murine TNF. Antitumor activity of rabbit TNF was shown against murine and human cancer cells in vivo and in vitro, and rabbit TNF was also capable of distinguishing malignant cells from normal cells.     

Expression of N-terminally truncated isoforms of CDP/CUX is increased in human uterine leiomyomas

Genetic analyses and mRNA expression studies have implicated CUTL1 as a candidate tumor-suppressor gene in uterine leiomyomas and breast cancers. However, modulation of CDP/Cux, the protein encoded by CUTL1, does not agree with this notion. The activity of CDP/Cux, which is the DNA binding subunit of HiNF-D, was upregulated as normal cells progressed into S phase and constitutively elevated in several tumor cell lines. Activation of CDP/Cux at the G(1)/S transition involved the proteolytic processing of the protein to generate a shorter isoform. Uterine leiomyomas represent a unique reagent for molecular analysis because they are resected as homogeneous tumor tissue together with the adjacent normal myometrium and they are often very large. In the present study, proteins were isolated from 16 pairs of matched tumors and adjacent myometrium and analyzed by Western blot and electrophoretic mobility shift assays. Strikingly, in 11/16 tumors, the steady-state level of small CDP/Cux isoforms was increased compared to normal control tissue. Where tested, a corresponding increase in CDP/Cux stable DNA binding activity was observed. DNA sequencing analysis of CUTL1 cDNAs from 6 leiomyomas, including 4 with LOH of CUTL1, did not reveal any gross rearrangement or point mutations. Altogether these findings suggest that CUTL1 is probably not the tumor suppressor on 7q22. Moreover, the frequent increase in smaller CDP/Cux isoforms indicates that molecular events associated with the truncation of CDP/Cux proteins may be selected in uterine leiomyomas.     

Identification of an anionic form of glutathione transferase present in many human tumors and human tumor cell lines

Glutathione transferase (GST) activity and GST isoenzyme composition have been determined for 24 human neoplasms and 6 human tumor cell lines. Substantial activity (40-1010 milliunits/mg protein) was identified in all tumor specimens examined and three of the tumor cell lines. Three tumor cell lines, the human small cell carcinoma line SW2-10S, the Burkitt's lymphoma derived cell line Raji, and the human breast carcinoma cell line MCF-7, contained minimal GST activity. Although the small size of the tumor samples precluded isoenzyme analysis by substrate specificities, analysis of GST activity following sample separation by isoelectric focusing indicated that the predominant (comprising at least 70% of the 1-chloro-2,4-dinitrobenzene-conjugating activity) GST isoenzyme in each of these primary tumor (17 of 17) and tumor cell line (3 of 3) extracts was anionic (isoelectric point, 4.5-4.8). In three tumor samples, adenocarcinomas of the lung, colon, and stomach, analysis by isoelectric focusing identified minor but detectable (10-20% of total) cationic GST. The anionic form of GST has been purified to homogeneity from three primary human tumors: a malignant melanoma; a mesothelioma; and a breast carcinoma. GST from these tumors consists of two subunits each of Mr 25,200. On Western blot analysis, antibodies raised against the anionic GST purified from mesothelioma detect protein of Mr approximately 25,000 in extracts of both normal kidney and tumors containing anionic GST activity but not in extracts of human liver that did not contain detectable anionic activity. The amino acid compositions of these proteins were quite similar to that previously described for GST-pi and the amino-terminal amino acid sequences for these tumor-derived isoenzymes are identical to one another and to that previously described for GST-pi from human placenta. GST is a major enzymatic activity in many human malignancies, comprising as much as 3% of the cytosolic protein of some tumors. Anionic GST is the predominant form of glutathione transferase activity in many human tumors and human tumor cell lines. In selected tumor samples the predominant anionic GST isoenzyme has been identified as a member of the pi class of this enzyme family. In addition, at least 3 of 17 tumor samples contained lesser but detectable amounts of cationic GST, probably of the alpha class. By conjugating glutathione with electrophilic anticancer drugs, the substantial levels of GST in human tumors may have a role in the innate or acquired resistance of these neoplasms to anticancer therapy.     

Enhanced expression of a 37,000-dalton protein in human lung cancers

When proteins in the tissue extracts of lung cancers were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), a 37,000-dalton protein (37K) was detected as a prominent form in all cases thus far examined. This protein was barely detectable or its amount was very small in the normal lung tissue extracts. Two-dimensional PAGE of lung tumor extracts showed 37K as a major spot with an isoelectric point of 7.6. An antibody against 37K was raised in a rabbit by injecting purified 37K. Immunoblot analysis of normal lung and lung tumor tissues revealed that the affinity-purified anti-37K antibody specifically recognized 37K and that the intensities of immunostaining corresponded to those of Coomassie brilliant blue staining.