Immune response against large tumors eradicated by treatment with cyclophosphamide and IL-12

Androgen has an important role in development of the prostate, and the actions of androgen are mediated, in part, by locally produced growth factors. These growth factors are postulated to mediate stromal-epithelial interaction in the prostate to maintain normal tissue physiology. Transforming growth factor-alpha (TGF-alpha) is one of the growth factors that can stimulate prostatic growth. The expression of TGF-alpha is thought to be regulated by androgen. The expression of epidermal growth factor receptor (EGFR), which is the receptor of TGF-alpha and EGF, also may be regulated by androgen. The hormonal and developmental regulation of TGF-alpha and EGFR messenger RNA (mRNA) levels in isolated epithelial and stromal cells from rat ventral prostate was investigated. The expression of mRNA for TGF-alpha and EGFR was analyzed by a quantitative RT-PCR (QRT-PCR) procedure developed. Observations from this assay demonstrated that both epithelial and stromal cells expressed the mRNA for TGF-alpha and EGFR. TGF-alpha mRNA expression was constant during postnatal, pubertal, and adult development of the prostate. EGFR mRNA expression was elevated at the midpubertal period and decreased with age. After castration of 60-day-old adult rats, both TGF-alpha and EGFR mRNA were significantly enhanced. TGF-alpha mRNA expression was stimulated by EGF in stromal cells (4.5-fold increase) but was not changed by any treatment in epithelial cells. EGFR mRNA levels were stimulated by EGF and keratinocyte growth factor treatment and inhibited by testosterone treatment in epithelial cells. Stromal cell EGFR mRNA levels were not affected by any treatment. Both testosterone and EGF stimulated incorporation of 3H-thymidine into prostatic stromal and epithelial cells. Anti-TGF-alpha antibody significantly inhibited testosterone-stimulated 3H-thymidine incorporation into stromal cells and epithelial cells. Immunocytochemical localization of TGF-alpha and EGFR demonstrated expression on the luminal surface of epithelial cells within prostatic ducts, and minimal expression was observed in stromal cells. Results indicate that testosterone does not directly regulate TGF-alpha mRNA levels but does inhibit EGFR mRNA levels. Interestingly, anti TGF-alpha antibody suppressed the effect of testosterone on 3H-thymidine incorporation into prostatic stromal and epithelial cells. This finding suggests that testosterone may act indirectly on prostatic cells to influence TGF-alpha actions. TGF-alpha mRNA levels were influenced by EGF in stromal cells only, and EGFR mRNA levels were influenced by testosterone, EGF, and keratinocyte growth factor in epithelial cells. These observations suggest that regulation of TGF-alpha and EGFR is distinct between the cell types. In conclusion, a network of hormonally controlled growth factor-mediated stromal-epithelial interactions is needed to maintain prostate development and function.     

Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse

Clara cells are primary targets for metabolically activated pulmonary toxicants because they contain an abundance of the cytochrome P450 monooxygenases required for generation of toxic metabolites. The factors that regulate bronchiolar regeneration after Clara cell injury are not known. Previous studies of naphthalene-induced bronchiolar injury and repair in the mouse have shown that epithelial cell proliferation is maximal 1 to 2 days after injury and complete 4 days after injury. Proliferation is followed by epithelial re-differentiation (4 to 14 days). In this study, mice were treated with the environmental pollutant naphthalene to induce massive Clara cell injury. The distribution and abundance of three growth-regulatory peptides (epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor (TGF)-alpha) was determined immunochemically during repair of this acute bronchiolar injury. EGFR and its ligands were detected at low levels in cells throughout the lung including peribronchiolar interstitial cells, blood vessels, and conducting airway epithelium. Immediately after naphthalene injury (1 to 2 days), EGFR, EGF, and TGF-alpha are expressed in increased abundance in squamous epithelial cells of the injury target zone, distal bronchioles. These immunopositive squamous cells are detected in clumps in the distal bronchioles at the time when cell proliferation is maximal. EGFR protein expression is decreased slightly 4 to 7 days after injury and continues to decrease below control levels of abundance 14 to 21 days after injury. This down-regulation of EGFR is not reflected in a corresponding decrease in EGF and TGF-alpha protein expression, indicating that control of cell proliferation is regulated at the receptor level. Co-localization of EGFR and bromodeoxyuridine-positive proliferating cells in the same bronchiole indicates that EGFR is up-regulated within the proliferative microenvironment as well as in specific proliferating cells within the injury target zone. The coincident localization within terminal bronchioles of EGFR, EGF, and TGF-alpha to groups of squamous epithelial cells 2 days after naphthalene injury suggests that these peptides are important in up-regulating cell proliferation after Clara cell injury in the mouse.     

Transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor expression in normal and diseased human adrenal cortex by immunohistochemistry and in situ hybridization

Because epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) have been implicated in the regulation of adrenocortical function, we used immunohistochemistry and in situ hybridization of EGF and TGF-alpha to study 41 specimens of human adrenal cortex, including 10 normal specimens, 15 aldosteronomas, five Cushing's adenomas, six adrenocortical incidentalomas, and five carcinomas to determine what role these growth factors play in controlling human adrenocortical function. Neither immunoreactivity nor mRNA hybridization signals to EGF was detected in any specimens, and EGF therefore may exert its effects on adrenal function as an endocrine hormone. TGF-alpha expression was detected at both protein and mRNA levels in normal and neoplastic adrenal cortex, demonstrating that TGF-alpha is synthesized locally in human adrenal cortex. TGF-alpha expression was observed in the cells with increased steroidogenesis, including compact tumor cells and zona fasciculata cells with lipid depletion, but did not necessarily correlate with production sites of any specific steroid hormone. EGFR immunoreactivity was more widely distributed than TGF-alpha immunoreactivity. Both TGF-alpha and EGFR expression were markedly elevated in adrenocortical carcinomas. TGF-alpha and EGFR thus appear to be involved in biological function in both normal and neoplastic human adrenal cortex. In addition, TGF-alpha and EGFR may play important roles in some biological features of adrenocortical malignancy.     

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.     

Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development

Mesenchymal-epithelial tissue interactions are important for development of various organs, and in many cases, soluble signaling molecules may be involved in this interaction. Hepatocyte growth factor (HGF) is a mesenchyme-derived factor which has mitogenic, motogenic and morphogenic activities on various types of epithelial cells and is considered to be a possible mediator of epithelial-mesenchymal interaction during organogenesis and organ regeneration. In this study, we examined the role of HGF during lung development. In situ hybridization analysis showed HGF and the c-met/HGF receptor gene to be respectively expressed in mesenchyme and epithelium in the developing lung. In organ cultures, exogenously added HGF apparently stimulated branching morphogenesis of the fetal lung. In contrast, HGF translation arrest or neutralization assays resulted in clear inhibition of epithelial branching. These results suggest that HGF is a putative candidate for a mesenchyme-derived morphogen regulating lung organogenesis. We also found that HGF is involved in epithelial branching, in collaboration with fibroblast growth factor (FGF) family molecule(s). In mesenchyme-free culture, HGF alone did not induce epithelial morphogenesis, however, addition of both HGF and acidic FGF (aFGF) or keratinocyte growth factor (KGF), ligands for the KGF receptor, induced epithelial branching more extensively than that was observed in explants treated with aFGF or KGF alone. In addition, the simultaneous inhibition of HGF- and FGF-mediated signaling using neutralizing antibody and antisense oligo-DNA resulted in drastic impairment of epithelial growth and branching. Possible interactions between HGF and FGFs or other growth factors in lung development is given consideration.     

Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells

Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium.     

Gut-trophic effects of keratinocyte growth factor in rat small intestine and colon during enteral refeeding

BACKGROUND: Keratinocyte growth factor (KGF) induces proliferation of gut epithelium in rat models, but KGF-nutrient interactions have not been studied. An experimental model of fasting-induced gut atrophy followed by different levels of enteral refeeding was used to investigate the influence of nutrient availability on the gut-trophic effects of exogenous KGF. METHODS: After a 3-day fast, rats were enterally refed either ad libitum or at 25% of ad libitum intake for 3 subsequent days. Either intraperitoneal KGF (5 mg/kg/d) or saline was given in each dietary regimen. Wet weight, DNA, and protein content were measured as indices of full-thickness cellularity in duodenum, jejunum, ileum, and colon. Villus height in small bowel segments and crypt depth in all gut tissues were measured as specific indices of mucosal growth. RESULTS: Refeeding at 25% of ad libitum intake significantly decreased full-thickness cellularity and mucosal growth indices in duodenum, jejunum, and ileum. In the colon, only protein content fell significantly and crypt depth was maintained. KGF administration during 25% refeeding did not alter full-thickness indices in any small bowel segment or affect jejunal mucosal growth. In contrast, KGF normalized duodenal villus height (p < .01) and duodenal and ileal crypt depth (p < .05) only in the 25%-refed model. KGF significantly increased ileal villus height in both ad libitum and 25%-refed rats (by 43% and 48%, respectively, p < .05) and markedly increased colonic cellularity and mucosal crypt depth with both levels of refeeding (p < .01). CONCLUSIONS: Rat small bowel growth is more sensitive than colon to the level of enteral refeeding after a 3-day fast. KGF administration does not affect jejunal growth, but specifically prevents atrophy of duodenal and ileal mucosa during hypocaloric, hyponitrogenous refeeding. In ileum and colon, some KGF-mediated growth responses are independent of the level of enteral refeeding. Thus gut-trophic effects of KGF and KGF interactions with the level of nutrient intake are tissue-specific.     

The heparin-binding domain of amphiregulin necessitates the precursor pro-region for growth factor secretion

The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.