MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer

Cancer-associated fibroblasts (CAFs) promote tumorigenesis, growth, invasion and metastasis of cancer, whereas normal fibroblasts (NFs) are thought to suppress tumor progression. Little is known about miRNAs expression differences between CAFs and NFs or the patient-to-patient variability in miRNAs expression in breast cancer. We established primary cultures of CAFs and paired NFs from six resected breast tumor tissues that had not previously received radiotherapy or chemotherapy treatment and analyzed with miRNAs microarrays. The array data were analyzed using paired SAM t-test and filtered according to α and q values. Pathway analysis was conducted using DAVID v6.7. We identified 11 dysregulated miRNAs in CAFs: three were up-regulated (miR-221-5p, miR-31-3p, miR-221-3p), while eight were down-regulated (miR-205, miR-200b, miR-200c, miR-141, miR-101, miR-342-3p, let-7g, miR-26b). Their target genes are known to affect cell differentiation, adhesion, migration, proliferation, secretion and cell-cell interaction. By our knowledge it is firstly identify the expression profiles of miRNAs between CAFs and NFs and revealed their regulation on the associated signaling pathways.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Membrane fluidity in normal and cystic fibrosis fibroblasts

We have observed distinct differences in the polarization of fluorescence and temperature dependent emission intensity of the highly fluorescent phospholipid derivative (1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)--aminocaproyl phosphatidylcholine (NBD-PC), when incorporated in the plasma membranes of normal and cystic fibrosis fibroblasts. Fluorescence polarization measurements indicate that the fluorochrome has a much higher degree of rotational mobility in cystic fibrosis fibroblasts as compared with normal cells. Temperature dependent transitions in the emission intensity of NBD-PC incorporated in normal fibroblasts are indicated at 17.7 and 21.2° C while the abnormal cell membranes apparently undergo transitions at 8.7 and 13.5° C. These differences might be due to changes in plasma membrane composition and/or organization, in the case of the cystic fibrosis cells.     

Proteoglycan synthesis in normal and Lowe syndrome fibroblasts

Lowe (oculocerebrorenal) syndrome (LS) is an X-linked disorder characterized by congenital cataracts, generalized hypotonia, mental retardation, and renal Fanconi syndrome. The basic defect remains unknown, but the possibility that fibroblasts express reduced sulfation of glycosaminoglycans has been studied in several laboratories. A mechanism involving overproduction of an enzyme (nucleotide pyrophosphatase) active against adenosine 3'-phosphate, 5'-phosphosulfate (PAPS) has been postulated. Decreased synthesis of normally sulfated glycosaminoglycans was also reported. We measured the synthesis of proteoglycans and glycosaminoglycans by incorporation of [3H]glucosamine and Na2(35)SO4 into cultured fibroblasts from four LS patients and related it directly to the synthesis in six normal fibroblast cultures. We found that the rate of synthesis varied greatly among the normal cultures (cv, 30%), but not significantly between LS and the normal. The LS fibroblasts' ability to sulfate glycosaminoglycans was assayed as the amount of 3H-glycosaminoglycan eluting at low ionic strength on anion exchange chromatography, the amount of non-sulfated disaccharide present in chondroitinase digests of labeled proteoglycans, and the ratio of 35S to 3H incorporation into proteoglycans. Each parameter suggested that the LS cells were synthesizing normally sulfated glycosaminoglycans (e.g. % delta Di-0S, 21 +/- 6 in normal; 27 +/- 6 in LS). The cells' ability to sulfate glycosaminoglycans was tested under conditions of markedly stimulated glycosaminoglycan synthesis, by treating the cultures with a beta-D-xyloside. LS and normal cells responded to the treatment by elevating the rate of synthesis of normally sulfated glycosaminoglycans (3.5-6-fold in normal, 3-7-fold in LS). Nucleotide pyrophosphatase activities were found to be elevated in each of our four LS cell strains as in the previous studies, excluding genetic heterogeneity as an explanation for our findings. We conclude that LS fibroblasts do not express defects in sulfation of glycosaminoglycans or in synthesis of proteoglycans.     

Cardiolipin induces premature senescence in normal human fibroblasts

Lipids seem to have various roles in cellular senescence. We found that cardiolipin very sensitively inhibits growth of normal human fibroblasts, whereas other phospholipids do not at 100 times higher concentrations. Growth arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated beta-galactosidase. Senescence markers such as the p21(waf1/sdi-1), fibronectin, and collagenase-I genes were significantly upregulated by cardiolipin. In addition, caldiolipin significantly increased in normally senesced human fibroblasts leaving other phospholipids unaltered. These results suggest that accumulation of cardiolipin is one of the causes for replicative senescence.     

Methionine biosynthesis in normal and transformed fibroblasts.

SV-40 transformed human fibroblasts show a growth requirement for methionine, whereas normal fibroblasts do not. Activities of the N⁵-methyltetrahydrofolate-homocysteine transmethylase and N^5–10-methylenetetrahydrofolate reductase in extracts of both cell lines are similar. These observations indicate that the absolute growth requirement for methionine observed in these transformed cells does not necessarily involve a deficiency in enzymes related to methionine synthesis.     

Protein methylation in normal and cystic fibrosis fibroblasts

Human-cultured fibroblasts contain protein methylase activities. These activities were determined and the enzymatic products were identified after acid hydrolysis of the protein substrate for protein methylases I (arginine) and III (lysine) and by organic solvent extraction of the methanol produced by alkaline treatment of the protein substrate (for the protein methylase II). A methylation of histidine residues of proteins occurs in cultured fibroblasts. Protein methylase activities were unmodified in the cystic fibrosis fibroblasts as compared to the control cells.     

Glutathione in fibroblasts from normal and cystinotic children

Intracellular levels of GSH and GSSG were compared in fibroblasts initiated from skin biopsies from normals and cystinotics. The concentration of GSH was essentially the same in both cell lines. However, the intracellular GSSG in cystinotic cells averaged less than half of that in normal cells. The ratio of intracellular GSH:GSSG was 10.6 in cells from cystinotics as compared with 4.7 in cells from normals. A significant distortion in the ratio of GSH:GSSG serves as another marker of the derangement of sulfur-amino acid metabolism in cystinosis. Whether this effect is primary or secondary remains to be elucidated.     

Adhesive specificity in normal and transformed mouse fibroblasts

Adhesive specificity was studied in normal and transformed $\text{Balb}\text{c}$ mouse fibroblasts by comparing the number of labeled cells collected from a suspension of these cells by aggregates of various cell types. Aggregates of the two malignant cells examined collected either very many cells (aggregates of SV3T3 cells) or very few cells (aggregates of 3T12 cells). In addition, the relative adhesive behavior of these two aggregate types did not vary according to the cell suspension in which they were circulated. These data make it unnecessary to assume that malignancy is always accompanied by a decrease in intercellular adhesion.The adhesive behavior of normal 3T3 cell aggregates, compared to the aggregates composed of either malignant cell type, varied according to the type of cells in the suspension. Aggregates of 3T3 cells collected an appreciable number of SV3T3 cells but few 3T12 cells. Collection of 3T3 cells by 3T3 aggregates was also low if the 3T3 cells of the suspension were harvested from confluent cultures. However, collection of 3T3 cells by 3T3 aggregates increased significantly, as compared to collection by SV3T3 and 3T12 aggregates in the same cell suspension, if the 3T3 suspension was prepared from sparse cultures.Flat-revertants of SV3T3 cells were also studied. These cells behave like nonmalignant 3T3 cells rather than like the SV3T3 cells from which they were derived.We suggest that malignancy may not be caused by decreased intercellular adhesion as compared to normal cells but, perhaps, by decreased intercellular recognition.     

Taurine uptake by normal and cystic fibrosis fibroblasts

Taurine deficiency recently has been proposed to be clinically significant in cystic fibrosis (CF). Uptake of [14C]taurine by four cystic fibrosis (CF) and three control fibroblast lines was examined to determine whether a generalized defect in taurine transport could contribute to the deficiency. The time course of uptake was linear up to 20 h and was similar in both CF and control fibroblasts. Taurine was avidly retained after uptake, and the effect of metabolic (chlorpromazine) and competitive (hypotaurine, L-leucine) inhibitors was similar in both CF and control cells. In contrast, while taurine uptake in a calcium-free medium was impaired in both CF and control fibroblasts, the impairment was significantly less in CF cells. The findings suggest that a generalized abnormality in taurine transport is unlikely to be responsible for the taurine deficiency in CF.     

Biosynthesis of sphingomyelinase in normal and Niemann-Pick fibroblasts

Deficient sphingomyelinase activity and massive storage of sphingomyelin are common to two clinically different forms of Niemann-Pick disease, called types A and B. Polyclonal antisera to human sphingomyelinase precipitated both enzyme activity and the polypeptide chain of purified placental sphingomyelinase. In normal fibroblasts, following a 19-h labelling period with [35S]methionine and immunoprecipitation of the labelled proteins, sphingomyelinase occurred as a single polypeptide with a mean molecular mass of 110 kilodaltons (kDa). Niemann-Pick disease type A and B fibroblasts also synthesized a sphingomyelinase polypeptide having the same molecular mass as that found in normal fibroblasts. In I-cell disease fibroblasts, a reduced amount of cross-reacting material was detected, suggesting that sphingomyelinase may be targeted to the lysosome via the phosphomannosyl receptor. Pulse-chase experiments demonstrated sphingomyelinase processing, as judged by a substantial loss of radiolabel and the appearance of an 84-kDa intermediate form of the enzyme. These results confirm and extend previous work based on autopsy specimens and urine, and show that Niemann-Pick disease fibroblasts synthesize a sphingomyelinase polypeptide. We show for the first time that an 84-kDa processed form of the enzyme is biosynthetically related to the 110-kDa polypeptide.     

Spreading-independent growth of normal fibroblasts in three-dimensional cultures

Changes of cell shape resulting from cellular flattening on culture substratum have previously been demonstrated to correlate with mitotic activity of normal animal cells in monolayer cultures. Here, we compared the shapes and proliferation of chick embryo fibroblasts cultured either in multicellular, multilayered sheets extended between glass fibres, or in standard monolayers. Fibroblasts in sheets retained the mitotic activity characteristic of that observed in sparse monolayer cultures, i.e. considerably higher that in confluent monolayers. Morphometric analyses revealed, however, that the cells in sheets were considerably less flattened than in monolayer cultures. These observations indicate that the modulation of culture conditions resulting in multidirectional cell stretching leads to the dissociation of flattening and mitotic activity of normal animal cells, so long as an intracellular stress field, generated by contractile cytoskeleton and stabilised by intercellular contacts, is maintained.     

Delayed chromosome changes in gamma-irradiated normal and Li-Fraumeni fibroblasts

Knockout mice with only one Trp53 allele (+/- genotype) are highly susceptible to radiation-induced cancers, possibly through numerical chromosome changes. Patients with the Li-Fraumeni syndrome, having heterozygous TP53 germline mutations (+/mut genotype), are also susceptible to spontaneous and radiogenic cancers. We have investigated the susceptibility of six Li-Fraumeni syndrome +/mut and six normal fibroblast strains to induced numerical and unstable structural aberrations at six population doublings after exposure to 3 or 6 Gy gamma rays. Four of the irradiated Li-Fraumeni syndrome strains showed small increases in both aberration types, similar to those seen in the normal strains. In two irradiated Li-Fraumeni syndrome strains, there were high levels of induced structural changes, and one of these showed a modest increase in hyperploidy. We suggest that enhanced sensitivity to delayed radiation-induced chromosome changes in Li-Fraumeni syndrome cells requires other genetic alterations in addition to TP53 heterozygosity, apparently in contrast to the situation in Trp53 heterozygous null mice. If such additional alterations occur in vivo in Li-Fraumeni syndrome patients, they may predispose them to radiogenic cancers, mainly through enhanced structural rather than numerical chromosome changes. Our findings raise questions about the validity of quantitative extrapolation of cytogenetic data from Trp53-defective mice to radiogenic cancer risk in humans.     

Study of thiol proteases of normal human skin fibroblasts

The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.     

Heme content of normal and porphyric cultured skin fibroblasts

Partial deficiencies in enzyme activities of the heme biosynthetic pathway have been demonstrated in cultured skin fibroblasts and other tissues from patients with protoporphyria (PP) and acute intermittent porphyria (AIP). We have quantitatively and qualitatively assessed the heme and free porphyrin content in cultured PP, AIP, VP (variegate porphyria, in which an enzymatic deficiency has not been identified), and normal skin fibroblasts during routine culture conditions in order to assess the overall metabolism of heme in these cells. The total heme concentration was not significantly different between control and porphyric lines; 189 +/- 15 pmoles/mg protein (mean +/- SEM) in controls, 154 +/- 17 in PP, 175 +/- 20 in AIP, and 181 +/- 81 in VP. The hemoprotein difference spectra were similar in all lines. Free porphyrins were not detected in any of the disorders. Despite partial deficiencies in enzyme activities of the heme pathway, porphyric fibroblasts thus maintain normal heme content during routine culture conditions without detectable porphyrin accumulation.