Clinical relevance of transforming growth factor alpha, epidermal growth factor receptor, p53, and Ki67 in colorectal liver metastases and corresponding primary tumors

To determine whether the expression of transforming growth factor alpha (TGF-alpha), its receptor (epidermal growth factor receptor [EGFr]), p53 nuclear protein, and proliferation influences prognosis of patients with liver metastases, a study was performed in 45 liver metastases and 33 corresponding primary colorectal carcinomas in patients referred for liver surgery. The expression of TGF-alpha, EGFr, p53 nuclear protein, and proliferation rate was correlated with clinicopathological characteristics and survival after partial liver resection. In liver metastases, TGF-alpha expression was low in 42%, intermediate in 35%, and high in 23%. TGF-alpha expression was higher in liver metastases derived from lymph node-positive primary carcinomas, in synchronous and in irresectable liver metastases compared with those derived from lymph node-negative primary carcinomas, metachronous, and resectable liver metastases. Nuclear p53 expression was found in 83% of primary tumors and 71% of liver metastases. p53 expression did not correlate with the various clinicopathological characteristics. Ki67 expression was not associated with clinicopathological characteristics in primary and metastatic tumors. In the 38 patients in whom a partial liver resection was performed, median survival was 25 months in patients with a higher TGF-alpha expression in the metastasis than in the primary tumor and 60 months in patients with comparable or lower TGF-alpha expression in the metastasis than in the primary tumor (P = .036). Median survival after liver resection was 21 months in patients with p53-negative liver metastases and 58 months in patients with p53-positive metastases (P = .043). By multivariate analysis, p53 and EGFr expression on liver metastases were the best predictors of disease-free survival after partial liver resection, with relative risks of 2.38 and 3.33, respectively. In patients with colorectal liver metastases, referred for liver surgery, a higher TGF-alpha expression is associated with unfavorable tumor characteristics, whereas p53 and absence of EGFr expression is associated with a better survival after partial liver resection.     

Regeneration of transplanted human liver: gene expression at the time of graft implantation

The liver tissue mRNA expression of protooncogenes c-fos, c-myc, c-Ha-ras, c-met and c-erb B1, and TGF alpha and TGF beta genes is sequentially and temporarily increased in the early stages after partial hepatectomy, ischaemia or other mitogenic stimuli. These gene expressions were studied in 38 samples of liver tissue from 24 patients who underwent orthotopic liver transplantation, at different evolution stages. Eleven samples were obtained from surgical liver biopsies before graft implantation at day 0 (Group A), 14 samples from percutaneous liver biopsies during the post-operative period from day 9 to day 48 (Group B) and 13 samples in the long-term follow-up period from day 102 to day 1,382 (Group C). Gene expression was studied using 32P-labeled cDNA and oligonucleotidic probe hybridization in slot blots. A GAPD gene was used as a control gene. All expression values of protooncogenes were related to those of the GAPD gene. After cold ischaema, the relative gene expression (quantity of specific mRNA/quantity of GAPD mRNA ratio) tended to diminish in most cases. The relative expressions of c-fos, c-myc, c-Ha-ras, c-met and TGF alpha gene were correlated at day 0. During and after the liver transplantation, an overexpression of c-fos, c-myc, c-Ha-ras, c-met and TGF alpha was observed in different pathological conditions such as cold ischaemia and conservation injury, acute rejection, and cytomegalovirus infection. In three cases the relative expression values of c-fos, c-myc and c-Ha-ras increased over long-term follow-up without any associated acute pathology. These results suggest the necessity of an intercellular mediation--by means of graft reperfusion--in induction of hepatocyte proliferation and regeneration of the transplanted liver.     

The localization of transforming growth factor alpha and epidermal growth factor receptor in stromal and epithelial compartments of developing human prostate and hyperplastic, dysplastic, and carcinomatous lesions

To gain insight into autocrine/paracrine mechanisms that may influence normal and abnormal growth of the human prostate, we studied the immunohistochemical localization of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFr) in fetal, neonatal, prepubertal, and young adult glands. Results were compared with findings in specimens of benign prostatic hyperplasia (BPH), dysplasia (prostatic intraepithelial neoplasia--PIN), and carcinoma. EGFr was strongly and exclusively expressed in fetal basal cells, whereas TGF-alpha was localized in these and secretory cells as well as in differentiating smooth muscle cells. In neonatal and prepubertal glands, EGFr continued to be found only in basal cells, whereas TGF-alpha was now present in smooth muscle and infrequently in secretory cells. In the normal adult prostate, the receptor was strictly localized in basal cells and in the lateral plasma membranes of secretory cells, whereas its ligand was exclusively expressed in smooth muscle. This pattern persisted in PBH, but both EGFr and TGF-alpha staining appeared to be enhanced in their respective cellular compartments. Irrespective of grade, in dysplasia diffuse-moderate EGFr and strong TGF-alpha staining were both present in a majority of secretory cells. Similarly, most cells in Gleason grade 3 and 4 carcinomas expressed both EGFr and TGF-alpha. Our findings suggest that an unregulated paracrine mode of growth attends the development of BPH, whereas malignant transformation and progression involves autocrine/paracrine mechanisms reminiscent of those found in the developing prostate.     

TGF-alpha, EGF, and their cognate EGF receptor are co-expressed with desmin during embryonic, fetal, and neonatal myogenesis in mouse tongue development

The developing mouse tongue provides a model for discrete patterns of morphogenesis during short periods of embryonic development. Occipital somite-derived myogenic cells interact with cranial neural crest-derived ecto-mesenchymal cells to form the musculature of the tongue. The biochemical signals that control close range autocrine and/or paracrine signaling processes required to establish the fast-twitch complex tongue musculature are not known. The present study was designed to test the hypothesis that desmin, epidermal growth factor (EGF), and transforming growth factor-alpha (TGF alpha) and their cognate receptor, epidermal growth factor receptor (EGFr), are co-expressed during tongue myogenesis and define specific developmental stages of tongue muscle cell differentiation. To test this hypothesis, we performed studies to analyze the timing, position, and concentration of desmin, TGF alpha, EGF, and EGFr from embryonic day 9 (E9) through birth in Swiss Webster mouse tongue development. Desmin, TGF alpha, EGF, and EGFr co-localized to cells of myogenic lineage in the four occipital somites and subsequently in myoblasts and myotubes from E9 through E17. By newborn stage, desmin is localized to discrete regions in myofibers corresponding to Z-line delimiting sarcomeres, and A-band within sarcomeres; immunostaining for desmin, TGF alpha, and EGF persisted in differentiated myotubes and striated skeletal muscle. Desmin increased from 0.01% at E11 to 0.51% of the total protein by E17 and at birth. Concomitantly, the patterns and increases in TGF alpha, EGF, and EGFr showed significant increases during the same developmental period. The temporal and positional co-localization of TGF alpha, EGF, and EGFr support the hypothesis that autocrine and paracrine regulation of desmin by actions of growth factor ligand and receptor defines critical stages of tongue myogenesis.     

Immunohistochemical analysis of 1,25-dihydroxyvitamin D3 receptor in cervical carcinoma

The immunohistochemical localization and expression of 1,25-dihydroxyvitamin D3 receptors (VDR) has been investigated in normal human cervical tissue (n = 15) and in cervical carcinomas (n = 23). VDR immunoreactivity (monoclonal antibody 9A7gamma) was compared with the staining patterns of transglutaminase K, cytokeratin 10 and Ki-67 in these tumours. Moderate to strong nuclear immunoreactivity for VDR was detected in almost all cervical carcinomas analysed. VDR staining was homogeneous, with no visual differences between individual tumour cells. Some 60% of normal cervical tissues revealed weak immunoreactivity for VDR. In normal cervical tissue, nuclear VDR staining was confined to the lower cervical layers, predominantly to the basal cell layer. Both the intensity of VDR immunostaining and the number of VDR-positive cells were up-regulated in cervical carcinomas compared with normal cervical tissue. No visual correlation was found for the coexpression of VDR with markers of proliferation and differentiation. Our findings indicate that: (1) cervical tissue may be a new target organ for therapeutically applied vitamin D analogues; (2) VDR is up-regulated at the protein level in cervical carcinomas compared with normal cervical tissue; (3) up-regulation of VDR in cervical carcinoma is induced not exclusively by alterations in epithelial differentiation or proliferation, but by different, unknown mechanisms; and (4) calcitriol and new vitamin D analogues exerting fewer calcaemic side-effects may be promising new drugs for the treatment or chemoprevention of metastasizing cervical carcinomas as well as of cervical precancerous lesions.     

Activation of erbB2 and c-src in phorbol ester-treated mouse epidermis: possible role in mouse skin tumor promotion

In recent work we showed that the EGF receptor (EGFr) was activated in tumor promoter treated mouse epidermis (Cell Growth & Differentiation, 6: 1447-1455, 1995). In the present study, we have investigated the possible role of other erbB family members in the process of tumor promotion. Both erbB2 and erbB3, but not erbB4, were expressed in cultured mouse keratinocytes and in mouse epidermis in vivo. In cultured mouse keratinocytes, EGF stimulated rapid tyrosine phosphorylation of erbB2 followed by a time-dependent degradation of erbB2 protein. Furthermore, an increase in erbB2:EGFr heterodimer formation was also induced by EGF. In contrast to the results with erbB2, EGF did not induce tyrosine phosphorylation, the degradation of erbB3, or erbB3:EGFr heterodimer formation in cultured keratinocytes. Further analyses revealed that c-src kinase activity was dramatically elevated in cultured mouse keratinocytes exposed to EGF. In mouse epidermis following multiple treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), the phosphotyrosine content of erbB2 was significantly elevated in a dose-dependent manner. Concomittantly, erbB2:EGFr heterodimer formation and c-src kinase activity were also elevated in TPA-treated epidermis. Structure-activity relationships with several phorbol ester analogs showed that the elevated phosphorylation of erbB2 in mouse epidermis followed closely with tumor promoting ability. Activation of erbB2 and c-src kinase were also observed in the epidermis of TGF alpha transgenic mice where expression of human TGF alpha was targeted to basal keratinocytes with the human K14 promoter. Collectively, the current data suggest that the activation of erbB2 in phorbol ester treated skin can be explained solely by a mechanism involving elevation of EGFr ligands and activation of the EGFr. In addition, activation of c-src may be an important downstream effector in mouse keratinocytes both in vivo and in vitro, following activation of the EGFr, erbB2, or both.     

A non-bile duct origin for intestinal crypt-like ducts with periductular fibrosis induced in livers of F344 rats by chloroform inhalation

To evaluate the toxic effects of prolonged exposure to chloroform vapors, female and male F344 rats were exposed to 0, 2, 10, 30, 90 and 300 p.p.m. chloroform by inhalation for 7 or 5 days/week for up to 13 weeks. The purpose of this study was to characterize a lesion that occurred in the livers of rats in the 300 p.p.m. exposure groups. Atypical glandular structures lined by intestinal-like epithelium and surrounded by dense connective tissue occurred in the livers of rats exposed to strongly hepatotoxic atmospheric concentrations of chloroform. Bile duct bromodeoxyuridine labeling indices as well as observations of the locations of the early lesions at the 3 and 6 week time points indicate that these lesions arose from a population of cells remote from the bile ducts. We refer to these lesions as intestinal crypt-like ducts with periductular fibrosis to distinguish them from true cholangiofibrosis. Here, intestinal crypt-like ducts with periductular fibrosis were seen only in rats exposed to 300 p.p.m. chloroform, and the multiplicity and severity of the lesions were greater in the right liver lobe. The lesion only occurred in association with liver necrosis and dramatic increases in hepatocyte labeling indices, while labeling indices in bile ducts in the same animals were not significantly different from controls. There was a treatment-related increase of transforming growth factor-alpha immunoreactivity in hepatocytes, bile duct epithelium, bile canaliculi and oval cells, and an increase in transforming growth factor-beta immunoreactivity in hepatocytes, bile duct epithelium and intestinal crypt-like ducts. Thus, intestinal crypt-like ducts with periductular fibrosis appeared to develop from a population of cells unrelated to bile ducts. Also, they occurred only in animals exposed to chloroform concentrations that induced significant hepatocyte necrosis and regenerative cell proliferation and were associated with increased growth factor expression or uptake.     

A centromeric satellite DNA of the unisexual Bacillus atticus (Insecta: Phasmatodea)

To investigate the relationship between dysplasia and carcinoma of the esophagus, 159 cases of esophageal carcinoma without any preoperative treatment were reviewed retrospectively. There were 75 dysplastic lesions in 32 cases (20.1%). The incidence of co-existence of dysplastic lesions was 0, 58.3, 31.3, 20.8 and 11.4% in intra-epithelial, mucosal and submucosal cancers and those invading the proper muscular layer and adventitia, respectively. Thus, excluding the cases of intra-epithelial carcinoma, the less advanced the lesion, the higher the incidence of dysplasia. Epithelial dysplastic lesions were classified as 12 with mild, 33 with moderate and 30 with severe degrees of dysplasia. Although the continuity of dysplastic lesions to the areas of carcinoma was not so frequent (48.0%), it was more often encountered in severe dysplasia rather than in moderate or mild dysplasia, which suggested some relationship between the severity of dysplasia and carcinoma. In the cases with a dysplastic lesion the multiplicity of squamous cell carcinoma and the intra-epithelial spread of the main lesion were more frequently seen (P < 0.001), suggesting a multicentric occurrence of dysplastic lesions and carcinomas.     

The syndrome of primary aldosteronism: a case study

OBJECTIVE: Marked alterations occur in the synthesis of endometrium-specific proteins during the first third of pregnancy in the baboon. Because epidermal growth factor (EGF) expression has been associated with proliferation in the human and mouse endometrium, we hypothesized that EGF, transforming growth factor-alpha (TGF alpha), and EGF receptor (EGF-R) expression in baboon endometrium may be modulated by the early invasive trophoblast and play a role in decidualization of the endometrial stroma. METHODS: Endometrial tissue was obtained from cycling baboons (n = 4-5 per time point), ovariectomized steroid-treated baboons (n = 4 per group), or from pregnant baboons on days 18-60 of pregnancy (n = 2-4 per group). The tissue was fixed in Bouin's solution and embedded in paraffin for immunocytochemistry using polyclonal antibodies against EGF and EGF-R and a monoclonal antibody to TGF alpha. RESULTS: Endometrial staining was located almost entirely in the glandular epithelium for TGF alpha and EGF-R in the follicular phase animals, whereas EGF staining was strongest in the periglandular stroma. In the luteal phase, specific staining for EGF also was detected in the glands as well as the periglandular stroma. There appeared to be little difference in endometrial staining between the late follicular and mid-luteal phase for TGF alpha and EGF-R. A similar pattern was observed in the steroid-treated animals. In the endometrium from pregnant animals, EGF, TGF alpha, and EGF-R intensely stained the glandular epithelium on days 18, 25, and 32. Both EGF and EGF-R showed light stromal staining on days 18 and 25. Light stromal TGF alpha staining was present on day 25 and became moderately intense by day 32. By day 60, the most intense staining for EGF and EGF-R was stromal. Staining of TGF alpha continued to be strong in the remaining epithelium through day 60. In placenta, EGF and EGF-R intensely stained the syncytiotrophoblast, but not the cytotrophoblast, whereas TGF alpha stained only the villous cytotrophoblast and intermediate cytotrophoblast within maternal blood vessels. There appeared to be no change in this staining pattern or intensity in the placenta throughout early pregnancy. CONCLUSIONS: This study demonstrates the presence of EGF, TGF alpha, and EGF-R in the endometrium during the cycle and early pregnancy. The detection of EGF, TGF alpha, and EGF-R in the stromal cells during pregnancy correlated with the onset of decidualization. We propose that EGF, TGF alpha, and EGF-R may play a role in glandular development during the cycle and in decidualization and implantation during early pregnancy.     

Epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor localization in the baboon (Papio anubis) oviduct during steroid treatment and the menstrual cycle

OBJECTIVES: Polypeptide growth factors may modulate the actions of estrogen (E2) and progesterone (P) in reproductive tissues in an autocrine/paracrine manner. The objective of this study was to determine whether the baboon oviduct contains epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and EGF receptor (EGF-R) and whether changes in their expression are correlated with various hormonal states. METHODS: Oviductal tissue was obtained from adult female baboons (Papio anubis) after oophorectomy and steroid treatment, and during the menstrual cycle. Ampullary regions were fixed in Bouin's fixative and embedded in paraffin for immunocytochemistry using rabbit polyclonal antibodies against EGF and EGF-R, and mouse monoclonal antibody against TGF alpha. RESULTS: Both EGF and EGF-R were present in all tissue compartments (most strongly in the epithelium, followed by smooth muscle and stroma) at all reproductive stages and showed similar staining patterns. However, the most intense immunoreactive product was found in the tissue obtained from the E2-treated and late follicular phase animals. At this time, intense staining was present in the apical regions of the mature ciliated cells, whereas the stain was dispersed uniformly over the cytoplasm of all other cell types. Immunoreactive TGF alpha was limited primarily to the nonciliated epithelial cells, and staining was most intense in the E2-treated and late follicular phase tissues. Transforming growth factor-alpha formed intense perinuclear deposits in the mature secretory cells, an area that corresponds to the Golgi region. No immunoreactive product was observed for any of these proteins when preimmune serum was substituted for the primary antibody or when the primary antibody was preabsorbed with antigen. CONCLUSION: In summary, EGF, TGF alpha, and EGF-R are present in the ampulla of the baboon oviduct. Moreover, the localization and intensity of immunoreactive product are dependent on cell type and hormonal state. These data are consistent with the concept that EGF, TGF alpha, and EGF-R may be regulated by E2 and P and thus may play a role in cell differentiation and function. In addition, the specific localization of TGF alpha suggests that this growth factor may be synthesized for release from the secretory cells and thus may also function as a modulator of gamete/embryo viability and development.     

Autonomous growth in serum-free medium and production of hepatocellular carcinomas by differentiated hepatocyte lines that overexpress transforming growth factor alpha 1

Transforming growth factor alpha (TGF-alpha) is a polypeptide closely associated with hepatocyte proliferation in vivo and in vitro. In order to investigate the mechanisms by which TGF-alpha contributes to hepatocyte replication and transformation, we isolated hepatocytes from mice bearing a human TGF-alpha transgene and examined their growth properties and gene expression in defined, serum-free culture. The transgenic hepatocytes continued to overexpress human TGF-alpha mRNA and peptide, and were able to proliferate without exogenous growth factors in primary culture, in contrast to nontransgenic mouse hepatocytes. In short-term culture the transgenic hepatocytes underwent 1 wave of DNA replication at 72-96 h in culture before senescing, similar to nontransgenic hepatocytes supplemented with epidermal growth factor. Constitutive expression of TGF-alpha rendered the transgenic hepatocytes unresponsive to further growth stimulation by exogenous TGF-alpha, as well as other mitogens such as epidermal growth factor and hepatocyte growth factor. However, it did not alter their sensitivity to growth inhibition by TGF beta 1, 2 and 3. The addition of nicotinamide to the culture medium enabled both transgenic and epidermal growth factor-supplemented normal hepatocytes to replicate repeatedly and survive for > or = 2 months in primary culture while maintaining differentiated traits. From these long-term primary cultures of transgenic and nontransgenic hepatocytes, we established immortalized cell lines (designated TAMH and NMH lines, respectively). Both lines continued to express differentiated adult hepatocytic markers such as albumin, alpha-1-antitrypsin, transferrin, and connexin 26 and 32 mRNAs, but also expressed mRNAs for the oncofetal markers alpha-fetoprotein and insulin-like growth factor II. Unlike the near-diploid NMH hepatocyte line, the transgenic TAMH hepatocyte line was quasi-tetraploid, strongly expressed human TGF-alpha mRNA, and was highly tumorigenic in nude mice. Well-differentiated hepatocellular carcinomas developed in nude mice given injections of the TAMH line, and these appeared similar to the primary liver tumors seen in TGF-alpha transgenic mice with regard to histology and strong expression of mouse and human TGF-alpha, insulin-like growth factor II, and alpha-fetoprotein mRNAs. Our data show that TGF-alpha overexpression causes autonomous hepatocyte proliferation and contributes to neoplasia but that additional cellular alterations must occur for carcinogenesis. Inappropriate expression of insulin-like growth factor II may constitute one of these steps. The TGF-alpha transgenic mouse hepatocyte line TAMH appears to undergo transformation in a similar manner to that of hepatocytes overexpressing TGF-alpha in vivo, and should serve as an ideal system in which to study hepatocarcinogenesis.     

Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), cripto-1, amphiregulin and epidermal growth factor receptor (EGFR) was studied in 51 premenopausal human ovaries at various phases of the menstrual cycle. Localization of mRNA for TGF alpha and EGF was also studied by in-situ hybridization. Immunoreactive TGF alpha was observed predominantly in theca cells in 12 of 33 antral follicles in the follicular phase (6/14 dominant follicles, and 6/19 non-dominant) but not in any of the 18 follicles in the luteal phase or in primordial and pre-antral follicles. TGF alpha immunoreactivity was present predominantly in the luteinized granulosa cells in 13 of 15 corpora lutea in the luteal phase, which are considered to be active in steroidogenesis, but not in any of the regressed corpora lutea. Accumulation of TGF alpha mRNA hybridization signal was observed only in the theca cells in the follicles and luteinized theca cells in the ovaries that were immunohistochemically positive for TGF alpha. EGFR immunoreactivity was detected in 24 of 33 antral follicles in the follicular phase and in two of 18 follicles in the luteal phase but not in any of the corpora lutea. Immunoreactive EGF, cripto-1 and amphiregulin or EGF mRNA was not detected in any follicles, corpora lutea, or the stroma cells examined. These results indicate that, of the epidermal growth factors examined in this study, TGF alpha is locally synthesized in normal cycling human ovaries and TGF alpha may be synthesized in theca cells and act on the granulosa cells in a paracrine fashion through the EGFR in ovarian follicles.     

DNA ploidy, P53 expression, and cellular proliferation in normal epithelium and squamous dysplasia of non-cancerous and cancerous human oesophagi

Ki67 expression, S-phase fraction, p53 immunoreactivity and DNA content were examined in morphologically normal mucosa and squamous dysplasia of both cancerous and non-cancerous human oesophagi in order to understand possible early events in the development of esophageal squamous cell carcinoma. 103 different foci from cancerous esophagi including 17 non-pathological epithelium, 10 mild, 17 moderate and 15 severe dysplasia, 14 intraepithelial carcinomas and 30 invasive squamous cell carcinomas were examined. Also studied were 57 biopsy specimens from cancer-free individuals, including 12 normal epithelia, 15 oesophagitis, and 16 mild, 11 moderate and 3 severe dysplasia. Areas of squamous dysplasia from both cancer-free and cancerous oesophagi were morphologically indistinguishable and both demonstrated increased cellular proliferation compared to normal or non-pathological epithelia. However, squamous dysplasia in cancerous oesophagi demonstrated significantly larger ki67 labelling indices and smaller S-phase fractions than dysplasia in cancer-free patients. Squamous dysplasia in cancerous and non-cancerous oesophagi demonstrated an non-diploid DNA histogram in 67.9% and 43.3% respectively. However, dysplasia from cancer-free individuals demonstrated a non-diploid pattern with one or more peaks (Type I non-diploid histogram) and that from oesophageal cancer patients predominantly exhibited non-diploid histograms without any distinctive peaks (Type II non-diploid histogram). Significant differences in the frequency of p53 positive foci were observed between dysplasia of cancer-free (23.3%) and cancerous (56.8%) oesophagi. IN cancerous oesophagi, dysplasia associated with Type II non-diploid histograms had a significantly larger number of p53-positive foci than those with diploid histograms or Type I non-diploid histograms. These results indicated that the biological features of squamous dysplasia were different between cancerous and non-cancerous human oesophagi despite indistinguishable morphological features. In addition, the combination of p53 immuno-histochemistry and DNA ploidy analysis may contribute to identify possible high-risk squamous dysplasia of the oesophagus.     

Immunohistochemical localization of transforming growth factor alpha in the major salivary glands of male and female rats

The three major salivary glands of normal male and female Fischer 344 rats of different ages were examined for the localization of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) by immunohistochemical staining. EGF was demonstrated only in the granulated convoluted tubule (GCT) cells of the submandibular gland, the results confirming the previous reports, and most abundantly in adult males and pregnant females. TGF alpha stain was localized in all three glands and was found throughout the entire duct system, excluding acinar cells. The myoepithelial cells of the sublingual gland were also reactive with the TGF alpha antibody. The specificity of the staining was confirmed by negative staining reaction with the absorbed antibody and by radio-immunoassay and Western blot methods. This is the first report describing the presence of TGF alpha in the rat salivary glands.     

Potential application of p53 as an intermediate biomarker in Barrett's esophagus

Diagnosis of the neoplastic progression in Barrett's esophagus using the histologic classification of dysplasia is frequently difficult. The tumor suppressor protein p53, when mutated, confers a promoter effect on cell growth. The purpose of this study was to evaluate the applicability of p53 as an intermediate biomarker of malignancy in Barrett's esophagus. Archival analysis of 100 biopsy specimens of Barrett's esophagus and 10 esophageal adenocarcinomas were compared with 35 chronic esophagitis biopsy specimens. Immunocytochemistry using an anti-p53 monoclonal antibody was performed and elevated immunoreactivity quantitated microscopically. Data were analyzed using a logistic regression model. Significant p53 immunoreactivity occurred as follows: chronic esophagitis (0%), Barrett's esophagus without dysplasia (10%), with low-grade dysplasia (60%), with high-grade dysplasia (100%), and adenocarcinoma (70%). All cases of Barrett's esophagus were significantly immunoreactive when compared with the chronic esophagitis cases (p = 0.001). There was an increase in p53 immunoreactivity as the histologic classification progressed toward adenocarcinoma (p = 0.001). Progression to high-grade dysplasia may be predicted based on p53 immunoreactivity. These findings suggest a role for p53 as an intermediate biomarker in Barrett's esophagus.     

Pharmacokinetics and pharmacodynamics of candesartan after administration of its pro-drug candesartan cilexetil in patients with mild to moderate essential hypertension--a population analysis

Among the peptide growth factors active in breast glandular cell proliferation epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are thought to play a major role in tumour development. They operate through binding to and activation of a common membrane receptor, defined as EGF-R. Their production is modulated by hormones and local growth factors. After it was shown by previous investigation in this laboratory that EGF-R could be detected in 90% of the tumours, but was masked by endogenous ligand in 36% of them, the question was raised as to the level of the ligand's expression in tumour tissue biopsies. Therefore, we investigated the expression of EGF and TGF alpha mRNA in 146 breast cancer biopsies by slot blot analysis using specific 32P-labelled probes. The data were correlated with sex steroids and EGF receptor content. Our results showed that EGF and TGF alpha coexisted in all tumour samples, and that their level of mRNA expression was similar in half of the tumours. Northern blot and polymerase chain reaction (PCR) analysis validated these findings. A significant direct correlation was found between the level of TGF alpha/EGF mRNA expression and the ER/progesterone receptor (PGR) content. TGF alpha and EGF mRNA levels were significantly higher in ER+ (P = 0.0015 and P = 0.0001, respectively) and in PGR+ tumours (P < 0.005 and P = 0.0001) than in their negative counterparts. Moreover, TGF alpha mRNA expression negatively correlated with the number of EGF-R binding sites measured by the standard method (P = 0.02), and it was significantly related to the number of sites occupied by endogenous ligand. In conclusion, it was shown that TGF alpha and EGF mRNA were coexpressed in all the tumour biopsies tested and that their level was higher in the hormone receptor positive than in negative samples. The correlation between the presence of ER/PGR sites, high level of TGF alpha/EGF mRNA and EGF-R occupancy by endogenous ligand is in favour of ER mediated control of TGF alpha and EGF production.     

The effects of growth factors on human normal placental cytotrophoblast cell proliferation

The effects of growth factors were investigated on the proliferation of a normal placental cytotrophoblast cell line (NPC). Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin-like growth factor-I (IGF-I) stimulated NPC cell proliferation. In contrast, TGFbeta1 was found to be a negative regulator, inhibiting EGF-induced cell proliferation. When EGF/TGF alpha receptor was analysed by radio-ligand binding, two binding sites of different affinities were revealed in the proliferating NPC cells but only the low affinity binding site was detected in the non-proliferating cytotrophoblast cells in primary cultures. The results suggest that EGF stimulates cytotrophoblast proliferation through high affinity binding sites.