Giulia Colletta

Expression of messenger RNA for amphiregulin, heregulin, and cripto-1, three new members of the epidermal growth factor family, in human breast carcinomas

The expression of amphiregulin (AR), heregulin (HRG), and cripto-1 (CR-1) mRNA transcripts was assessed in 60 human primary breast carcinoma. AR and HRG transcripts were expressed respectively in 58% and 25% of the carcinomas as measured by Northern blot analysis. CR-1 mRNA was found in 77% of the carcinomas using Reverse Transcriptase-PCR analysis. Coexpression of two or three of these peptides was observed in several specimens. There was no significant association between AR, HRG, and CR-1 expression and nodal status, EGF receptor, or c-erbB-2 protooncogene expression in these tumors. However, a significant association between AR expression and estrogen receptor positivity was observed.

Cyclin D1 and cyclin E expression in malignant thyroid cells and in human thyroid carcinomas

Evidence of the involvement of cyclin gene alterations in human cancer is growing. In this study, we sought to determine the pattern of expression of cyclin D1 and cyclin E in normal and malignant thyroid cells. Quiescent rat thyroid cells in culture, induced to synthesize DNA by thyrotropin (TSH), expressed cyclin D1 gene after 6 hr and cyclin E gene with a peak at 18 hr from the stimulus; K‐ras‐transformed rat thyroid cells, which grew without addition of hormones necessary for normal cell proliferation, expressed elevated levels of cyclin D1 and cyclin E, compared with normal differentiated thyroid cells. Human benign and malignant thyroid tumors and their relative normal tissues were then analyzed. Neither major genetic alterations nor amplifications for cyclin D1 and cyclin E genes were found by Southern blot analysis in genomic DNAs extracted from all types of thyroid tumors. Moreover, statistical analyses of densitometric values from Northern blots did not show increased levels of cyclin D1 and E mRNAs in the tumor samples, compared with normal thyroid. Immunohistochemical analyses of formalin‐fixed, paraffin‐embedded sections of tissues with specific antibodies revealed a prevalent cytoplasmic cyclin E staining in the thyroid tissues analyzed. Cyclin D1, instead, was present in the cytoplasm of normal thyroids and adenomas, but in 31% of thyroid papillary carcinomas analysed, it was overexpressed, with a localization in the nucleus. Our in vivo observations suggest that unlike cyclin E, elevated nuclear cyclin D1 expression defines a subset of thyroid papillary carcinomas, and might be a contributory factor to thyroid tumorigenesis. Int. J. Cancer 76:806–811, 1998.© 1998 Wiley‐Liss, Inc.

Overexpression of transforming growth factor β-type II receptor reduces tumorigenicity and metastastic potential of K-ras-transformed thyroid cells

Expression of type II receptor of transforming growth factor beta (TbetaRII) is necessary for this factor to inhibit the growth of thyroid epithelial cells. In rat thyroid transformed cells, the resistance to transforming growth factor beta (TGFbeta) is associated with a decreased expression of TbetaRII mRNA and protein. Reduced TbetaRII expression has also been found in human thyroid differentiated and undifferentiated carcinomas. To investigate the role of TbetaRII in modulating the tumorigenic potential of k-ras-transformed thyroid cells, we transfected these cells with an expression vector carrying the human TbetaRII gene, regulated by an inducible promoter. Isolated clones, overexpressing TbetaRII, showed a reduction in the anchorage-dependent and -independent cell growth, compared with control k-ras-transformed cells. When transplanted in athymic nude mice, the transfected clones presented a decrease in tumorigenicity with respect to the highly malignant parental cells. Moreover, the diminished tumorigenic ability of the clones studied was accompanied by a statistically significant reduction in spontaneous and lung artificial metastases. Taken together, our data demonstrate that TbetaRII acts as a potent tumor suppressor gene when overexpressed in malignant thyroid cells.

Restored expression of transforming growth factor β type II receptor ink-ras-transformed thyroid cells, TGFβ-resistant, reverts their malignant phenotype

Transforming growth factor β1 (TGFβ1) inhibits the growth of normal rat epithelial thyroid cells (FRTL‐5 strain) by counteracting thyrotropin (TSH)‐stimulated DNA synthesis and by slowing the cells in the G1 phase of the cell cycle. Here, we have studied two clones of FRTL‐5 thyroid cell line transformed by the wild type (wt) v‐k‐ras oncogene (K.M.A1, K.M.A2) and one clone (A6) transformed by a temperature‐sensitive (ts) v‐k‐ras mutant. Anchorage‐dependent as well as anchorage‐independent growth of these k‐ras‐transformed cells was not inhibited by TGFβ1. TGFβ1 resistance appeared to be dependent by a functional p21 k‐ras, because A6 cell growth was partially inhibited at the nonpermissive temperature (39°C). To determine the basis for TGFβ1 resistance in k‐ras‐transformed thyroid cells, we looked for possible defects in the expression of type I (TβR‐I/ALK5) and type II TGFβ receptors (TβR‐II). Lower levels of type II receptors were present in all of the k‐ras‐transformed clones, as revealed by both Northern blot and cross‐linking experiments.

Thyrotropin stimulates transcription from the ferritin heavy chain promoter

Thyrotropin (TSH) is the primary hormone regulating the activity of the thyroid gland. We have recently shown that TSH stimulates H-ferritin mRNA levels in rat thyroid. Ferritin plays a key role in determining the intracellular fate of iron. The induction of ferritin synthesis by iron in liver is regulated both at transcriptional and translational levels. Here we present evidence that the mechanisms by which TSH regulates the mRNA levels are mediated by a diffusible product acting in trans on its own promoter. In fact, the H-ferritin promoter mediates increased CAT activity in response to hormone induction. Our results identify transcription as an important regulatory step of TSH action. They suggest that TSH induces expression of the ferritin gene, and that continuous protein synthesis is required to maintain basal ferritin gene expression in the absence of hormone.

Interferon effects on Friend leukaemia cells. I. EXpression of virus and erythroid markers in untreated and dimethyl sulphoxide-treated cells.

The effects of low doses (40 to 1000 units/ml) of mouse interferon (IF) on the expression of Friend leukaemia virus (FLV) and globin genes in Friend leukaemia cells (FLC) have been examined. IF blocks production of extracellular virus, but virus antigens accumulate in the cytoplasm. In cells treated with IF at the time of seeding, there is a reduction in the amount of RNA specified by the lymphatic leukaemia virus (LLV) component of FLV; with the same IF dose there is a small but definite stimulation of haemoglobin and globin mRNA synthesis. The effects of IF on LLV gene expression are even more pronounced in dimethyl sulphoxide (DMSO)-stimulated LFC. No correlation was found between LLV gene expression and the appearance of erythroid markers.

Enhanced expression of heregulin in c‐erb B‐2 and c‐Ha‐ras transformed mouse and human mammary epithelial cells

Heregulin β1 was found to stimulate the anchorage‐dependent, serum‐free growth of nontransformed human MCF‐10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor α, or amphiregulin, heregulin β1 was also able to induce the anchorage‐independent growth of MCF‐10A cells. In contrast, the anchorage‐dependent, serum‐free growth of c‐Ha‐ras or c‐erb B‐2 transformed MCF‐10A cells was unaffected by heregulin β1, whereas heregulin β1 was able to stimulate the anchorage‐independent growth of these cells. c‐Ha‐ras or c‐erb B‐2 (c‐neu) transformed MCF‐10A or mouse NOG‐8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell‐associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF‐10A cells and transformants also express c‐erb B‐3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha‐ras or erb B‐2 transformed mammary epithelial cells.

TSH is able to induce cell cycle-related gene expression in rat thyroid cell

Rat thyroid cells in culture (FRTL-5 strain) require thyrotropic hormone (TSH) for growth. TSH alone in serum free medium is able to induce DNA synthesis of FRTL-5 cells. DNA synthesis occurs 18-20 hours following TSH stimulation of quiescent cells. Here we demonstrate that two sets of genes, related to the entry of cells in the S phase, are induced by TSH: 1) immediate early genes, such as c-jun and a gene coding for a zinc-finger protein Xrox 20/Egr2, both having a pattern of expression similar to the c-fos oncogene; 2) early delayed genes such as ornithine decarboxylase (ODC), 2F-1, a gene that shows a strong similarity in aminoacid sequence to a mitochondrial ADP/ATP carrier, and the asparagine synthetase gene (TS11). Furthermore, an increased expression of the histone H3 gene, a typical marker of S phase, has been observed in TSH-treated FRTL-5 cells.

Heregulin-dependent autocrine loop regulates growth of K-ras but not erbB-2 transformed rat thyroid epithelial cells.

The EGF‐like family of proteins, such as epidermal growth factor (EGF), transforming growth factor α (TGFα), amphiregulin (AR), betacellulin (BTC), cripto‐1 (CR‐1), and heregulin (HRG), plays an important role in the pathogenesis of several human carcinomas as autocrine growth factors. Differentiation and proliferation of rat thyroid cells in culture (FRTL‐5 cells) are regulated by thyrotropin (TSH); withdrawal of TSH from culture medium produces growth arrest, whereas its addition to quiescent cells stimulates cell entry into S phase. Instead, transformed thyroid cell lines as FRTL‐5H2 cell line, overexpressing erbB‐2, Kimol cells, transformed by the wild‐type K‐ras and A6 clone, transformed by a temperature sensitive K‐ras mutant, can grow without addition of TSH to the culture medium. In order to identify whether EGF‐like growth factors and corresponding receptors (erbB‐2, erbB‐3, and erbB‐4) could be involved in the autonomous growth of these transformed rat thyroid epithelial cells, Northern blot for mRNA analysis and Western blot for protein expression were performed. In contrast to normal control FRTL‐5 cells, both K‐ras and erbB‐2‐transformed cells expressed elevated levels of erbB‐2 receptor. Moreover, both K‐ras transformed cells, Kimol and A6 cells, but no FRTL‐5H2 cells, were found able to express also high levels of erbB‐4 receptor and HRG/NDF ligand. Treatment of K‐ras transformed thyroid cells with neutralizing antibody against HRG/NDF reduced by 50% cell proliferation. These data indicate that unlike the erbB‐2 overexpressing FRTL‐5 cells, in K‐ras rat thyroid epithelial cells, the growth factor heregulin signals through the heterodimer erbB‐2/erbB‐4 receptors in an autocrine fashion. J. Cell. Physiol. 176:383–391, 1998.

TGFβ inhibits rat thyroid cell proliferation without alterations in the expression of TSH-induced cell cycle-related genes

Transforming growth factor beta (TGF beta) is a secreted polypeptide factor that is thought to play a major role in the regulation of proliferation of many cell types and various differentiation processes. TGF beta acts on thyroid cells by inhibiting cell proliferation and expression of differentiation markers, such as thyroglobulin production and iodide uptake. Exponentially growing thyroid cells cultures accumulate mostly in G0/G1 after exposure to TGF beta for 48 hours. TGF beta inhibits the TSH induced transition of quiescent thyroid cell from the G0 to the S phase. These effects on the thyroid cell growth, however, are not mediated by changes in the TSH-induced cell cycle-related genes expression; both immediate early and progression genes expression is unaffected by the TGF beta treatment.