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D G Beer

Publications and source records attributed to D G Beer.

At least 55 records · Page 3Linked to original sources
PubMed1993

Finite proliferative lifespan in vitro of a human breast cancer cell strain isolated from a metastatic lymph node.

We recently described culture conditions that allow proliferation of metastatic human breast cancer cells from biopsy specimens of certain patient samples. These conditions resulted in the development of an immortalized cell strain designated SUM-44PE. These same culture conditions were used to isolate a human breast cancer cell strain from a metastatic lymph node of a separate breast cancer patient. The SUM-16LN human breast cancer cells isolated from this specimen were cultured either in serum-free medium or serum-containing medium supplemented with insulin and hydrocortisone. Unlike the SUM-44PE cells that have proliferated in culture continuously for over two years, SUM-16LN cells proliferated in culture for approximately 200 days and underwent 15 to 20 population doublings before undergoing cell senescence. No cells of this strain proliferated beyond passage 8. SUM-16LN cells were keratin-19 positive and had an aneuploid karyotype. These cells overexpressed p53 protein and had an amplified epidermal growth factor (EGF) receptor gene that resulted in high level expression of tyrosine phosphorylated EGF receptor protein. Despite the presence of high levels of tyrosine phosphorylated EGF receptor in these cells, they proliferated in serum-free, EGF-free medium and did not secrete detectable levels of EGF-like mitogenic growth factor. In addition, these cells were potently growth inhibited by all concentrations of exogenous EGF tested and by the neutralizing EGF receptor antibody Mab 425. These results suggest that the high level of tyrosine phosphorylated EGF receptor present in these cells is the direct result of receptor overexpression and not the result of the presence of a stimulatory ligand. Thus, SUM-16LN represents a human breast cancer cell strain that exhibited genetic and cellular characteristics of advanced human breast cancer cells. Nevertheless, these cells exhibited a finite proliferative lifespan in culture, suggesting that cellular immortalization is not a phenotype expressed by all human breast cancer cells.

Blotting, Western↗
PubMed1993

Sucrase-isomaltase gene expression in Barrett's esophagus and adenocarcinoma.

BACKGROUND: Specialized Barrett's esophageal mucosa, characterized by incomplete intestinal metaplasia of the esophageal mucosa, is associated with the development of adenocarcinoma. Although the intestinal disaccharidase sucrase-isomaltase (SI) has been shown in incomplete intestinal metaplasia of the stomach, it is commonly believed that Barrett's mucosa does not express SI based on the lack of enzymatic activity. This study was undertaken to determine whether the SI gene is expressed in Barrett's epithelium and its associated adenocarcinoma at the level of messenger RNA (mRNA) and protein. METHODS: Reverse transcription polymerase chain reaction was used to determine the presence of SI mRNA in Barrett's esophagus and esophageal adenocarcinomas. Cellular localization of SI protein was determined by immunohistochemistry. RESULTS: SI mRNA was identified in 76% of Barrett's epithelium and 82% of esophageal adenocarcinomas. The transcriptional initiation site for SI in these tissues was identical to that of the small intestine. Immunohistochemical localization showed that SI was directed to the apical membrane in Barrett's epithelium in contrast to a more diffuse cytoplasmic pattern in esophageal adenocarcinomas. CONCLUSIONS: Columnar cells of specialized Barrett's epithelium express SI and are, therefore, phenotypically similar to those in incomplete intestinal metaplasia of the stomach with respect to intestinal gene expression.

Adenocarcinoma↗
PubMed1992

Decreased hepatic phosphoenolpyruvate carboxykinase gene expression after 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment: implications for the acute toxicity of chlorinated dibenzo-p-dioxins in the rat.

Decreased activity of the rate limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), has been recently suggested to be the critical lesion in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We now show that other toxicologically relevant chlorinated dibenzo-p-dioxins (CDDs), with chlorine substituents in the crucial 2-,3-,7-, and 8-positions, exert the same effect on PEPCK activity. The doses required to cause this enzyme inhibition are within the acutely toxic range for each homologue, suggesting the same mechanism of action for these compounds. To further investigate the mechanism whereby dioxins decrease PEPCK activity, Northern analysis was performed using a cDNA probe complementary to a portion of the PEPCK mRNA. We could demonstrate that after TCDD treatment hepatic PEPCK mRNA was decreased by as much as 90% compared to pair-fed control animals (day 8 after dosing). This decrease in PEPCK mRNA was paralleled by a decrease of the amount of PEPCK protein and enzymatic activity. These results indicate that the physiological changes which occur in TCDD-treated animals (decreased feed consumption, low plasma insulin and elevated plasma corticosterone levels) which under normal conditions increase PEPCK gene expression and enzyme activity, are not effective in stimulating PEPCK synthesis in TCDD-treated animals.

Animals↗
PubMed1990

Ki-ras activation and expression in transformed mouse lung cell lines.

The Ki-ras proto-oncogene is activated by specific point mutations and is the transforming gene often identified in rodent and human lung tumors. An in vitro model to aid in the study of the consequences of Ki-ras activation and expression in mouse lung is needed. Accordingly, we have examined cell lines derived from chemically induced mouse lung tumors as well as spontaneous transformants of untreated mouse lung epithelial cells. The specific Ki-ras-activating gene mutations and the level of mRNA expression were examined for each cell line. Polymerase chain reaction and oligonucleotide hybridization were used to demonstrate that five of seven transformed lung cell lines contain codon 61 Ki-ras-activating mutations, resulting in an arginine substitution for wild-type glutamine. One transformed line contained this activating mutation and had also lost, or contained an altered, wild-type codon 61 Ki-ras allele. No codon 12 Ki-ras mutations were observed. Two transformed and two nontransformed epithelial lung cell lines contained only the wild-type codon 12 and 61 Ki-ras alleles. Northern blot analysis demonstrated that the Ki-ras mRNA was present in all the cell lines and was overexpressed in some, but not all, of the transformed lung cell lines. Those transformed lines with the highest levels of Ki-ras mRNA also expressed more H4-histone mRNA, suggesting that these cells have a greater proliferation rate. The level of Ki-ras mRNA increased during the proliferation of the nontransformed lung cells but then decreased upon reaching confluency. In contrast, the level of Ki-ras mRNA in the transformed lung cells was high during both growth and confluency, suggesting a potential defect in the regulation of Ki-ras in these cells. These lung cell lines will help provide a better understanding of the regulation of both the Ki-ras proto-oncogene and oncogene in the lung.

Adenocarcinoma↗
PubMed1990

Specific Ki-ras codon 61 mutations may determine the development of urethan-induced mouse lung adenomas or adenocarcinomas.

In A/J strain mice, the carcinogen urethan induces lung adenomas and adenocarcinomas that contain Ki-ras-activating mutations primarily in codon 61. These mutations affect the middle adenine in codon 61 resulting in the substitution of either arginine (AT----GC transition) or leucine (AT----TA transversion) for the wild-type glutamine. To analyze the expression of the wild-type and mutant Ki-ras mRNAs in primary mouse lung tumors and transformed mouse lung cell lines, we utilized reverse transcription of total mRNA and DNA amplification by the polymerase chain reaction. The wild-type allele of codon 61 was expressed in all normal lung and primary tumor samples and in all transformed cell lines, except one. Significantly, the leucine-substituted allele was expressed primarily in very small lung adenomas, whereas the arginine-substituted allele was expressed in large lung adenocarcinomas and transformed lung cell lines. The relative amounts of expression of the mutant versus wild-type Ki-ras alleles and the total Ki-ras mRNA expression was similar in both lung adenomas and adenocarcinomas. Further, the arginine mutant allele was present in adenocarcinomas having either alveolar or papillary tumor morphologies. These results suggest that the specific activating Ki-ras mutation is more critical to either lung adenoma or adenocarcinoma development than is the tumor's cell of origin or the extent to which the mutant alleles are expressed. A distinct role of the specific activating Ki-ras mutations in affecting lung tumor growth or malignant potential is indicated.

Adenocarcinoma↗
PubMed1989

Proto-oncogene activation during chemically induced hepatocarcinogenesis in rodents.

The liver is a frequent site for the development of chemically induced cancer in rodents. This is primarily owing to the capability of the liver to activate a large variety of exogenous chemicals metabolically to reactive electrophilic species that can covalently interact with cellular DNA and other macromolecules (Miller and Miller, 1966; Miller, 1978). It is the potential alteration of the hepatocellular genome by mutational events that forms the theoretical basis for the heritable nature of cancer as well as, at least in part, the altered phenotype of neoplastic cells; however, our understanding of the exact nature of these heritable genetic alterations remains fragmentary. Within the last decade the delineation of the molecular basis of viral oncogenesis, especially by retroviruses, has revealed potential targets in the cell genome for the reactive forms of chemical agents in relation to their carcinogenic action (Bishop, 1987). Primary among such potential targets are proto-oncogenes, homologous to the transforming genes of oncogenic retroviruses from which they have evolved (Temin, 1974). The objective of this brief review is to consider the evidence that induced alterations in the structure and/or regulation of expression of proto-oncogenes may play one or more roles in rodent hepatocarcinogenesis, especially in relation to the stages of initiation, promotion, and progression (Pitot et al., 1988).

Animals↗
PubMed1988

Expression of the c-raf protooncogene, gamma-glutamyltranspeptidase, and gap junction protein in rat liver neoplasms.

Female Harlan Sprague-Dawley and F-344 rats were subjected to a 70% hepatectomy and 18 h later given one dose of 30 mg diethylnitrosamine/kg body weight. Beginning 1 week later, the animals were fed a diet containing 0.05% phenobarbital. Groups of rats were sacrificed 6 and 15 months later, and the livers were either frozen for cryostat sectioning or used to isolate RNA. Primary liver tumors present in these animals were used for RNA isolation, and a portion was taken for histopathological analysis. Eleven of 13 primary lesions, consisting of either neoplastic nodules or hepatocellular carcinomas, showed elevated levels of mRNA for the c-raf protooncogene. Increased c-raf mRNA in these tumors appeared to be unrelated to their cellular proliferative status inasmuch as the levels of c-raf mRNA did not correlate with levels of H4 histone mRNA. Decreased expression of the major rat liver gap junction protein mRNA was observed in all of the primary tumors. Immunocytochemical analysis using an anti-gap junction antibody revealed a decrease in gap junction immunoreactivity in some but not all preneoplastic focal lesions. All preneoplastic foci having positive gamma-glutamyltranspeptidase enzyme staining also exhibited a marked increase in gamma-glutamyltranspeptidase mRNA as determined by in situ hybridization. The possible relation of alterations of the mRNA levels of c-raf and the gap junction protein to the further development of preneoplastic foci is discussed.

Animals↗
PubMed1988

Gene expression during multistage hepatocarcinogenesis.

Several multistage models of hepatocarcinogenesis in the rat have now been developed. In one of these models at least three distinct stages--initiation, promotion, and progression--can be delineated. The first and last are irreversible, while that of promotion is reversible. Quantitation of each stage is possible. While several variable marker enzymes have been utilized to identify and characterize preneoplastic lesions during the stage of promotion, preliminary investigations indicate that the transcriptional activation of proto-oncogenes, particularly that of c-raf-1 proto-oncogene, may be used as an indicator of those preneoplastic lesions that will potentially develop into malignant neoplasms.

Animals↗
PubMed1987

Specific gene expression during compensatory renal hypertrophy in the rat.

The compensatory growth of the kidney which is induced by unilateral nephrectomy is a highly regulated process resulting principally in hypertrophy of the remaining kidney. The events which regulate this process are unknown. We have examined the levels of transcripts for the proto-oncogenes, myc, H-ras, K-ras, and fos, and the cellular genes, H4 histone, ornithine aminotransferase, and gamma-glutamyl transpeptidase, following unilateral nephrectomy in the rat. The pattern of expression of c-myc, c-H-ras, and c-K-ras during compensatory growth of the kidney differs from the pattern of expression of these proto-oncogenes during liver regeneration, in which, unlike the kidney, hyperplasia rather than hypertrophy predominates. The lack of change in the abundance of these proto-oncogene transcripts following unilateral nephrectomy suggests a primary relationship between the expression of these proto-oncogenes and DNA synthesis and indicates there may be separate signals for cell growth, one to double cell size and one to replicate DNA. Increased mRNA transcripts for the enzymes ornithine aminotransferase and gamma-glutamyl transpeptidase were induced in the contralateral kidney after nephrectomy. The time course of expression for these two enzymes differs. The early expression of the gamma-glutamyl transpeptidase gene may indicate an involvement of this glutathione-metabolizing enzyme during renal compensatory growth, while the function of the delayed increase in ornithine aminotransferase transcripts in the remaining kidney is not apparent.

Animals↗
PubMed1987

Biological markers characterizing the development of preneoplastic and neoplastic lesions in rodent liver.

The identification of biological "markers" indicating distinctively different functions between preneoplastic and neoplastic as compared with normal cells has been a subject of intensive investigation, especially as additional technology becomes available. Although no distinct single biochemical marker is ubiquitous to the process of neoplasia or even to a single histogenetic type of neoplasm, a variety of histogenetic types of neoplasms in the human and experimental animals exhibit an extreme degree of marker or phenotypic heterogeneity. Particularly well studied are markers which occurred during the process of hepatocarcinogenesis in the rodent as well as in its final product, the hepatoma. Although phenotypic heterogeneity is characteristic of hepatocellular carcinomas in both the rat and mouse, some degree of predominant marker pattern(s) has become apparent. In multistage hepatocarcinogenesis in the rat a frequent but not completely ubiquitous marker is the enzyme gamma-glutamyltranspeptidase. In the mouse, although such markers have not been as extensively studied as in the rat, glucose 6-phosphatase is a predominant but not exclusive histochemical marker. Many preneoplastic lesions occurring during the stage of promotion exhibit reduced levels of enzymes of oxidative xenobiotic metabolism, but this pattern is not ubiquitous. Studies on the transcription of specific genes in mouse liver as well as preneoplastic and neoplastic lesions in this tissue further demonstrate the phenotypic heterogeneity characteristic of differentiated hepatocellular carcinomas. In general, current evidence supports the two theses that no single biologic marker or set of markers is uniquely characteristic of the preneoplastical and/or neoplastic phenotype and marker or phenotypic heterogeneity is by far the rule rather than the exception in hepatocarcinogenesis in the rodent and quite possibly in all histogenetic types of neoplasms in mammals.

Albumins↗
PubMed1986

Expression of albumin messenger RNA detected by in situ hybridization in preneoplastic and neoplastic lesions in rat liver.

The process of chemical hepatocarcinogenesis is characterized by the appearance of preneoplastic lesions showing changes in the expression of various marker enzymes. We have analyzed the phenotype of small preneoplastic foci and expansively growing nodules in liver sections obtained from rats treated with various carcinogens. Changes within the lesions in canalicular adenosine triphosphatase, gamma-glutamyl transpeptidase, NADPH-(cytochrome P-450) reductase, cytochrome P-450 PB2, epoxide hydrolase, and glycogen content were detected by means of enzyme histochemical and immunohistochemical staining procedures. In parallel sections the expression of albumin messenger RNA was investigated by in situ hybridization using a 35S-labeled albumin specific complementary DNA probe. In general, small preneoplastic lesions showed unchanged levels of albumin messenger RNA. In contrast, the expression of albumin messenger RNA was found to be reduced to varying degrees in large hepatic nodules. An expression of alpha-fetoprotein messenger RNA could not be detected in any of the nodules. No direct correlation between the enzyme phenotype of the lesions and the degree in reduction of albumin messenger RNA could be established except that the reduction was most pronounced in nodules which had lost their ability to store glycogen. Since the synthesis and excretion of albumin is a typical function of the differentiated hepatocyte in the adult animal, the observed decrease in albumin messenger RNA expression in large hepatic nodules is in accordance with the hypothesis of a gradual dedifferentiation or retrodifferentiation of the cell population during carcinogenesis. Hyperplastic nodules produced by continuous treatment of rats with 4-dimethylaminoazobenzene showed increased rather than decreased albumin levels. The analysis of albumin messenger RNA expression might therefore be used as a tool to discriminate between nodules of differing biological nature and fate.

Adenosine Triphosphatases↗
PubMed1986

Expression of H-ras and c-myc protooncogenes in isolated gamma-glutamyl transpeptidase-positive rat hepatocytes and in hepatocellular carcinomas induced by diethylnitrosamine.

The appearance of gamma-glutamyl transpeptidase (GGT) in focal areas of hepatocytes is a widely used histochemical marker for the identification of preneoplastic cell populations. The characterization of these GGT-positive preneoplastic cells in relation to possible alterations in protooncogene expression may help define cellular changes occurring during the early stages of hepatocarcinogenesis. Female Sprague-Dawley rats were subjected to a two-thirds partial hepatectomy, followed 18 h later by a single intragastric administration of 30 mg of diethylnitrosamine per kg and subsequent feeding of a diet containing 0.05% phenobarbital for 6 or 11 mo. Primary cell suspensions were obtained after the perfusion of liver with collagenase. Cell debris and nonviable cells were removed with multiple washes and a Percoll gradient step. GGT-positive hepatocytes were enriched from the cell suspension by adherence to an affinity-purified GGT antibody affixed to Petri dishes. These dishes allowed the selective adherence and collection of up to 2.28 X 10(6) GGT-positive cells per liver. The starting cell population and the isolated GGT-positive and -negative cells were then used for subsequent analysis. RNA was prepared from the cell isolates and from hepatocellular carcinomas induced with the same diethylnitrosamine and phenobarbital regimen as that used to induce GGT-positive foci; 10 micrograms of total cellular RNA were used for Northern blot hybridizations. The blots were probed with 32P-labeled c-myc, H-ras, and albumin DNAs. The results indicate that GGT-positive hepatocytes do not differ from the other hepatocyte populations in either the size or amount of mRNA transcripts for the c-myc and H-ras protooncogenes. Increased expression of c-myc and H-ras was observed in some malignant lesions and may represent a secondary alteration occurring during the multistage process of hepatocarcinogenesis.

Albumins↗
PubMed1986

Genetic variation in the proliferation of murine pulmonary type II cells: basal rates and alterations following urethan treatment.

Susceptibility to urethan-induced pulmonary tumorigenesis varies among inbred strains of mice. A genetic basis for this variation was sought using three strains with widely differing tumor multiplicities after urethan treatment. Twenty-one mice from each of strains A/J (high susceptibility), BALB/cByJ (intermediate susceptibility; hereafter called cBy), and C57BL/6J (low susceptibility; hereafter called B6) were treated i.p. with 1 mg urethan/g body weight, and sacrificed at 0 (no urethan), 12, 24, 36, 48, 65, and 80 days after treatment (three mice per strain per time point). Each mouse was given 1 muCi [3H]thymidine/g body weight 45 min before sacrifice. Lungs were processed for autoradiography, and labeling indices were independently determined for non-tumor-associated type II cells and for tumor cells (most tumors arise from alveolar type II pneumocytes in A/J mice). Three categories of proliferative differences were found. First, statistically significant differences (P less than 0.05) among all strains were found for type II cell labeling indices in untreated mice, and these differences persisted for 65 days after urethan treatment. Proliferative rates were highest in A/J mice and lowest in B6 mice, while cBy mice were intermediate. Secondly, the peak of type II cell labeling occurred 12 days following urethan in strains A/J and cBy, but at 24 days in B6 mice. This difference is consistent with the fact that tumors were observed earlier following urethan treatment in A/J and cBy mice (at 36 days) than in B6 mice (at 48 days). Finally, the labeling indices in A/J and B6 tumors were high at first (6 and 4%) and then declined to 1-1.5% by 80 days after urethan treatment, while cBy tumor labeling indices remained at about 1.5% throughout the experimental period. These results suggest that the variation in susceptibility to urethan-induced lung tumorigenesis among different strains of mice is related to the normal basal rates of lung mitoses in these strains. Mice may be particularly sensitive to urethan during cell division, making strains with a higher rate of mitosis more susceptible to tumorigenesis.

Animals↗
PubMed1985

Genetic influence on type 2 or Clara cell origin of pulmonary adenomas in urethan-treated mice.

Urethan-induced lung adenomas arising from alveolar type 2 cells and from bronchiolar Clara cells have distinct histologic patterns; and examination of all lung adenomas found in A/J, SWR/J, BALB/cByJ, 129/J, and RIIIS/J inbred mice, 14-16 weeks after a single urethan injection, demonstrated that both tumor types were present in all five strains. The relative numbers of each tumor type varied significantly among the different strains. No correlation was observed between the type of tumor formed and tumor multiplicity. Augmentation of tumor multiplicity by chronic butylated hydroxytoluene (BHT) treatment following urethan injection did not change the proportion of the 2 tumors from that observed with urethan alone. This finding demonstrated that BHT stimulated the development of both tumor types equally. The tumors found 14 weeks after a single urethan injection into neonatal BALB mice were larger than those found 14 weeks after urethan injection into adult BALB mice, but the proportion of alveolar and papillary tumors was the same for both groups. Lymphocytic infiltration into tumors was mainly associated with Clara cell-derived tumors. The proportion of type 2 cell- and Clara cell-derived lung adenomas appeared to be controlled primarily by the genetic background.

Adenoma↗
PubMed1985

Changes in cyclic adenosine 3':5'-monophosphate-dependent protein kinases during the progression of urethan-induced mouse lung tumors.

The cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases in lung adenomas are functionally different from those of normal lung. The relevance of this change to neoplastic conversion was examined by comparing tumor kinases with those obtained from the normal cell of origin and by studying the kinases at different stages of tumor growth. Lung tumors were collected from A strain mice at different times after a single injection of urethan. These tumors are predominantly of alveolar type two cell origin, and cAMP-binding proteins in extracts from isolated type two cells and from lung adenomas at various stages of tumor progression were compared. Both the incorporation of the cAMP photoaffinity analogue, cyclic 8-azidoadenosine 3':5'-[32P]monophosphate (8-N3-[32P]cAMP), into the regulatory subunits of the type I (RI) and type II (RII) cAMP-dependent protein kinases and the autophosphorylation of RII were similar in extracts from whole normal lung and from type two cells. Altered protein kinases are thus not characteristic of normal type two cells. Lung tumors showed a decrease in photodetectable RII which correlated in degree with tumor size and extent of anaplasticity. This decreased RII photolabeling during tumor growth was associated with increased RII autophosphorylation. In contrast, decreased RII photolabeling in extracts from neonatal lung is accompanied by a substantial decrease in RII autophosphorylation. The characteristics of RII during normal development thus clearly differ from those during neoplastic development. An increase in the amount of an Mr 37,000 proteolytic fragment derived from R-subunits was also noted as a function of tumor progression. DEAE-cellulose chromatography of tumor cytosol showed that the increase in the amount of Mr 37,000 protein was accompanied by increased subunit dissociation of the type I isozyme. The dissociated RI subunit has been shown to be more sensitive to cleavage by a Ca2+-dependent neutral protease than when RI was in the holoenzyme form. This protease is present in both normal lung and lung adenomas, and its activity increases during the later stages of tumor progression. A comparison of cAMP binding and the light-induced covalent incorporation of 8-N3-[32P]cAMP showed that, for both RI and RII, photoincorporation was about 75% as efficient as noncovalent binding. In contrast, although the Mr 37,000 fragment can be photolabeled with low concentrations of 8-N3-[32P]cAMP, noncovalent cAMP binding to the endogenous Mr 37,000 fragment could not be demonstrated with a standard filtration assay. Such altered cAMP binding characteristics following Ca2+-dependent proteolysis of R-subunits would all

Affinity Labels↗
PubMed1984

Developmental changes in the endogenous Ca2+-stimulated proteolysis of mouse lung cAMP-dependent protein kinases.

Regulatory subunits (R subunits) of mouse lung cAMP-dependent protein kinases undergo age-dependent changes in endogenous proteolysis, with the greatest amount of the major Mr = 37,000 proteolytic fragment detectable during fetal and neonatal development. Homogenization of lung in the presence of various protease inhibitors does not affect this age-related difference, suggesting that the observed quantitative change in R subunit proteolysis occurs in vivo. Mechanisms were sought to account for this age-dependent change. The production of a Mr = 37,000 proteolytic fragment can be stimulated in lung extracts by the addition of exogenous calcium and is due to the action of an endogenous Ca2+-stimulated protease. Neonatal lung extracts show more Ca2+-stimulated proteolysis of R subunits than adult extracts, although only slight age-related differences in either the Ca2+-stimulated protease or its specific endogenous inhibitor were observed. Age-dependent differences in R subunits which may affect sensitivity to proteases were also examined. Analysis of the two-dimensional patterns of adult and neonatal 8-N3-[32P]cAMP-labeled R subunits before or after limited proteolysis with trypsin suggests that the R subunits are structurally similar. Differences are found, however, in the relative proportions of adult and neonatal Type I R subunits (RI) in the holoenzyme or dissociated forms. An increased proportion of neonatal R subunits exist in the dissociated state, whereas adult R subunits exist primarily in the holoenzyme form. Dissociated R subunits from mouse lung are more susceptible than the holoenzyme to limited proteolysis by the partially purified lung Ca2+-stimulated protease. Dissociation of the holoenzyme in vivo may be a major factor in the age-dependent proteolytic changes observed in mouse lung protein kinases.

Adenosine↗
PubMed1984

Autoradiographic localization of specific [3H]dexamethasone binding in fetal lung.

The cellular and subcellular localization of specific [3H]dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of [3H]dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Quantitative binding studies, involving incubation of intact tissue with competing ligand and subsequent subcellular fractionation, show this to be specific, nuclear binding characteristic of glucocorticoid receptors. Autoradiographs of [3H]dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of [3H]dexamethasone. Because of the known importance of the mesenchyme in controlling lung development and the ability of glucocorticoids to stimulate lung development, these results suggest that many of the growth-promoting effects of glucocorticoids may be mediated through the mesenchyme. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and [3H]dexamethasone binding, a relationship is observed between extensive mesenchymal [3H]dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.

Animals↗