Nancy L. Thompson

Transforming growth factor beta: biochemistry and roles in embryogenesis, tissue repair and remodeling, and carcinogenesis.

Transforming growth factor (TGF)- β plays essential roles in embryogenesis, particularly during periods of morphogenesis. Some of the same embryological mechanisms are reiterated in the adult during the normal processes of tissue remodeling and repair and aberrantly in various pathological processes, including carcinogenesis. This chapter highlights the new advances in the understanding of the complex biology of TGF- β and discusses the chemistry of TGF- β . The broad range of biological activities of TGF- β makes it highly likely that other peptide activities—purified by presumably novel and specific assays—will result from TGF- β once their amino acid sequence is determined. TGF- β 1 and 2 are two homologous forms of a homodimeric peptide with molecular weight of 25,000. Every chain of the peptide contains 112 amino acids of which nine are cysteine residues. The chapter reviews the structure of TGF- β 1 and 2 and TGF- β gene family. The biological activities of the members of the TGF- β family are described in the chapter. The chapter further reviews the regulation of gene activity by TGF- β, antibodies to TGF- β , and role of TGF- β in embryogenesis, tissue repair and remodeling, and carcinogenesis and other proliferative diseases.

Transforming Growth Factor Beta-1 in Acute Myocardial Infarction in Rats

TGF-beta 1 has been examined in the heart during myocardial infarction caused by ligation of the left coronary artery. Infarcted and uninfarcted myocardium have been compared by immunohistochemical staining of TGF-beta 1 and by Northern blot analysis of mRNA. Normal ventricular myocytes are strongly stained by an antibody to TGF-beta 1. Progressive loss of staining of these myocytes begins within 1 hr after coronary ligation. However, by 24-48 hr after ligation, intense staining of myocytes at the margin of infarcted areas is seen. Northern blots of infarcted myocardium 48 hr after ligation show a 3- to 4-fold increase in the principal 2.4 kb TGF-beta 1 mRNA; there is also a marked increase in a minor 1.9 kb transcript. In the same tissue samples, there is a 2-fold decrease in the mRNA for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase. The results indicate a significant role for TGF-beta in the response of the heart to injury.

Transforming Growth Factor-Beta: Possible Roles in Carcinogenesis

TGF-beta is the prototype of a large family of multifunctional regulatory proteins. The principal sources of the peptide, platelets and bone, suggest that it plays a role in healing and remodeling processes. In vitro, TGF-beta is chemotactic for monocytes and fibroblasts and can greatly enhance accumulation of extracellular matrix components by fibroblasts. Its ability to stimulate the formation of granulation tissue locally and the demonstration of specific time- and tissue-dependent expression in embryogenesis suggest that similar mechanisms are operative in vivo. By analogy to its effects in wound healing and embryogenesis, it is proposed that TGF-beta, secreted by tumour cells, can augment tumour growth indirectly by effects on the stromal elements. These effects include suppression of the immune response, and enhancement of both angiogenesis and formation of connective tissue. Many tumour cells have escaped from direct growth inhibitory effects of TGF-beta by a variety of mechanisms including inability to activate the latent form of the peptide, loss of cellular receptors for TGF-beta, and loss of functional intracellular signal transduction pathways.

Purification and Culture of Oval Cells from Rat Liver

Publisher Summary This chapter discusses the purification and culture of oval cells from rat liver. Oval cells are liver epithelial cells that proliferate during the early stages of hepatocarcinogenesis induced by many chemicals. The biological importance of oval cells derives from two observations: (1) in azo dye carcinogenesis, oval cells give rise to hepatocytes and to transitional cells, which combine morphological features of these two cell types, and at the early stages of hepatocarcinogenesis induced not only by azo dyes but also by other carcinogens, oval or transitional cells contain many of the markers associated with hepatic neoplasia. An important step in the assessment of the biological properties of oval cells was the identification of AFP in oval and/or transitional cells at the early stages of carcinogenesis and the conclusion that the rapid increase in serum AFP detected a few days or weeks after the administration of many carcinogens is due to the proliferation of oval cells. In the early stages of carcinogenesis, the proliferation of duct cells, transitional cells, or small hepatocytes takes place and these cell types form a conspicuous but heterogeneous oval cell compartment where duct cells most likely predominate. Transitional cells may mature into new diploid hepatocytes, which have been detected in hepatocarcinogenesis induced by ethionine and other chemicals. Such diploid hepatocytes or the transitional cells might be the populations from which liver tumors may develop several months later. Most aspects of this working hypothesis are testable experimentally and some of the work on the isolation and characterization of oval cells and the growth of oval cells in cultures leading to the formulation of the hypothesis.

Characterization and evaluation of detoxification functions of a nontumorigenic immortalized porcine hepatocyte cell line (HepLiu).

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TA1 (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.

Tightly regulated induction of the adhesion molecule necl-5/CD155 during rat liver regeneration and acute liver injury†

TuAg1/TagE4, the rat ortholog of the human poliovirus receptor CD155, is expressed on a high percentage of rat hepatocellular carcinomas. Recent studies have shown that TuAg1/TagE4/CD155 is a member of the nectin family of immunoglobulin (Ig)‐like cell adhesion molecules, designated necl‐5. Necl‐5 is present at exceedingly low levels in adult epithelial tissues but is upregulated in primary cultures of rat hepatocytes, suggesting that disruption of liver architecture triggers its expression. To explore this possibility, we examined expression of necl‐5 after two‐thirds partial hepatectomy or carbon tetrachloride (CCl4)–induced acute injury. Using quantitative real‐time polymerase chain reaction (QPCR), we found that necl‐5 mRNA levels increased 15‐fold by 9 hours, and decreased to 4‐fold above baseline by 24 hours after partial hepatectomy. Necl‐5 mRNA levels increased over 100‐fold 6 hours after treatment with CCl4, reaching a peak of 140‐fold above baseline by 10 hours, and thereafter rapidly declining. Necl‐5 was localized at the membrane of midlobular and centrilobular hepatocytes 10 to 48 hours after CCl4 exposure. Northern blot analysis demonstrated a close correlation between the kinetics of necl‐5 expression and the immediate–early response gene c‐myc. Subconfluent cultures of the non‐transformed liver epithelial cell line WB‐F344 expressed high levels of necl‐5, which was down‐regulated as cells approached confluence. The transformed WB‐F344 line GP7TB did not demonstrate density‐dependent regulation of necl‐5 expression. In conclusion, we report the in vivo induction of necl‐5 in rat hepatocytes and provide evidence that both necl‐5 mRNA and protein are tightly regulated in adult epithelial cells and tissue. (HEPATOLOGY 2006;43:325–334.)

Transforming Growth Factor-β: Multifunctional Regulator of Cell Growth and Phenotype

Transforming growth factor-/3 (TGF-/3) is the founder member of a growing family of structurally related peptides that are involved in the regulation of cell growth and development in organisms as phylogenetically distant as flies and man. TGF-/3 itself is a multifunctional molecule whose biological effects are highly context-dependent. Thus, depending on the cell type and the cell environment, TGF-/3 in vitro can stimulate or inhibit proliferation, promote or block differentiation, and modulate cellular function.’.’ Two closely related forms of TGF-/3 have been identified. Type 1 and type 2 TGF-/3 share 70% sequence identity and are indistinguishable in most biological assay systems. However, activities unique to cach type are emerging2 and it can be anticipated that the two types may be differentially regulated. Structurally, the active form is a disulfide-linked homodimer of 25 kDa and the monomeric unit is encoded as the C-terminal 112 amino acids of a 390-residue p r e c ~ r s o r . ~ With 2 mg extractable TGF-p/kg, platelets are the most concentrated source of TGF-/3 1 in the body, probably reflecting an important role for the molecule in wound healing4 Bone is also a major source (0.1 mg/kg), and all other tissues examined have detectable, though lower (0.01-0.03 mg/kg) TGF-/3 levels. Thus, although TGF-/3 was initially discovered and named in the context of a transformation assay, the ubiquitous distribution of the molecule suggests that it must play a fundamental regulatory role in normal cell physiology.

Adenoviral modulation of the tumor-associated system L amino acid transporter, LAT1, alters amino acid transport, cell growth and 4F2/CD98 expressionwith cell-type specific effects in cultured hepatic cells.

Altered expression of metabolite transporters is observed frequently in tumor cell lines and primary neoplasms. The extent to which these may to contribute to the growth autonomy associated with cancer is not clear. LAT1 is a major L‐type amino acid transporter over‐expressed in a variety of cancer types and a light chain component of the CD98 heterodimer. We utilized an adenoviral expression system to modulate the level of LAT1 in a hepatic in vitro model to examine phenotypic changes associated with short‐term exogenous and blocked expression. LAT1 levels were increased three fold and resulted in increased L‐type amino acid transport as a result of adenoviral expression in murine hepatocytes. The protein was expressed on the cell surface and complexed with the CD98 heavy chain known as 4F2. Surprisingly, levels of the total CD98 protein complex were increased 2.4‐fold as a result of adenoviral expression of light chain only, suggesting coordinate regulation. Exogenous overexpression was less effective in normal rat liver cells relative to mouse. LAT1 antisense expression in hepatic tumor cells resulted in a modest though statistically significant decrease in cell number, viability and S‐phase cells over a 5‐day period relative to controls despite the absence of a significant decrease in L‐type transport over this period. These studies are preparatory to in vivo efforts focusing on LAT1/CD98 as a potential therapeutic target.

Cell CAM 105 Isoform RNA Expression Is Differentially Regulated during Rat Liver Regeneration and Carcinogenesis

Cell CAM 105 (C-CAM) is a member of the carcinoembryonic antigen family and has been characterized as a rat hepatocyte cell-cell adhesion molecule via antibody activity. Two isoforms have been cloned and differ primarily in the length of the cytoplasmic domain. Despite extensive structural studies, little is known about their function and regulation in vivo. We have examined C-CAM expression during rat liver regeneration and hepatocarcinogenesis. Steady-state C-CAM RNA varied less than 3-fold during regeneration with subtle changes in the isoform ratio both before and after hepatocyte division. In liver tumors and transformed cells derived from tumors, however, large-scale decreases were observed in C-CAM RNA with wide variations in isoform ratios. In general, RNA decreases were reflected at the protein level. Our data suggest whereas down-regulation and alterations in C-CAM isoform ratio are transient during regulated liver growth, they are permanent in malignancy and may modulate hepatocyte adhesion.

Addressing the Challenge of Diversity in the Graduate Ranks: Good Practices Yield Good Outcomes

This paper describes practices designed to improve graduate student training outcomes in the sciences. It describes work to increase student diversity in the graduate ranks and documents the success of trainees. The practices designed to achieve these outcomes are broadly applicable to all graduate training programs and students.