Russette M. Lyons

TGF-β1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains

TGF-beta 1 is demonstrated to inhibit skin keratinocyte proliferation when added during the G1 phase of the cell cycle. Human foreskin keratinocytes transformed with either HPV-16 or -18 or SV40, however, were resistant to the growth inhibitory effects of TGF-beta 1. Since TGF-beta 1 appears to inhibit keratinocyte growth through down-regulation of c-myc, it was hypothesized that these DNA tumor viruses might be modulating the response to TGF-beta 1 via this pathway. Transient expression of proteins HPV-16 E7, adenovirus type 5 E1A, and SV40 large T antigen is demonstrated to block TGF-beta 1 suppression of c-myc transcription. This effect was not observed with DNA tumor virus transforming proteins mutated in their pRB binding domain. These observations indicate that pRB or another protein that interacts with this binding domain mediates TGF-beta 1 regulation of c-myc gene expression and growth inhibition.

The cell biology of transforming growth factor β

The TGF beta family of polypeptide growth factors regulates a remarkable diversity of cellular functions, many of which are not directly associated with cell growth. The present review has summarized many of the recent studies that have just begun to conceptually integrate this expanding array of TGF beta functions into the context of a three-dimensional, multicellular organ or tissue, be it normal or diseased. This fascinating research strongly implicates TGF beta as a key modulator of a wide variety of important physiologic and pathophysiologic processes.

Receptor interactions involved in adenoviral-mediated gene delivery after systemic administration in non-human primates

Adenovirus serotype 5 (Ad5)-based vectors can bind at least three separate cell surface receptors for efficient cell entry: the coxsackie-adenovirus receptor (CAR), alpha nu integrins, and heparan sulfate glycosaminoglycans (HSG). To address the role of each receptor involved in adenoviral cell entry, we mutated critical amino acids in fiber or penton to inhibit receptor interaction. A series of five adenoviral vectors was prepared and the biodistribution of each was previously characterized in mice. To evaluate possible species differences in Ad vector tropism, we characterized the effects of each detargeting mutation in non-human primates after systemic delivery to confirm our conclusions made in mice. In non-human primates, CAR was found to have minimal effects on vector delivery to all organs examined including liver and spleen. Cell-surface alpha nu integrins played a significant role in delivery of vector to the spleen, lung and kidney. The fiber shaft mutation S*, which presumably inhibits HSG binding, was found to significantly decrease delivery to all organs examined. The ability to detarget the liver corresponded with decreased elevations in liver serum enzymes (aspartate transferase [AST] and alanine transferase [ALT]) 24 hr after vector administration and also in serum interleukin (IL)-6 levels 6 hr after vector administration. The biodistribution data generated in cynomolgus monkeys correspond with those data derived from mice, demonstrating that CAR binding is not the major determinant of viral tropism in vivo. Vectors containing the fiber shaft modification may provide for a detargeted adenoviral vector on which to introduce new tropisms for the development of targeted, systemically deliverable adenoviral vectors for human clinical application.

Expression of and response to growth regulatory peptides by two human pancreatic carcinoma cell lines.

Two human pancreatic adenocarcinoma cell lines (PANC 1 and MIA PACA 2) were examined for expression of growth factors that could potentially play a role either in growth regulation of the tumor cells, or in cells that comprise the stromal elements of tumors. Both cell lines expressed transforming growth factor-α (TGFα), basic fibroblast growth factor (bFGF), c-sis (PDGF B chain), TGFβ1, and TGFβ3 mRNA by Northern blot analysis. Only the PANC 1 cells, however, expressed the TGFβ2 transcript. TGFP-like competing activity was found in medium conditioned by either cell line, but TGFα-like [epidermal growth factor (EGF)-competing] activity was not detected in the medium from either cell line by radioreceptor assay. TGFα and EGF caused concentration-dependent stimulation of soft agar colony growth of the MIA PACA 2 cells, while only TGFα caused a significant but less dramatic stimulation of soft agar growth of the PANC 1 cells. Insulin stimulated the anchorage-independent growth of MIA PACA 2 but not PANC 1 cells. Likewise, bFGF also caused a concentration-dependent stimulation of MIA PACA 2 but not PANC 1 growth in soft agar, and PDGF had no effect on the growth of either cell line. TGFβ had no inhibitory or stimulatory effect on soft agar colony growth of either the PANC 1 or the MIA PACA 2 cells, although both cell lines exhibited high affhity, saturable TGFβ binding sites, and TGFβl was capable of autoinduction of TGFβ1 mRNA expression in PANC 1 cells. The ability to continue to respond to positive growth regulatory factors coupled with the loss of responsiveness to negative growth factors may be important in the pathogenicity of these aggressive tumors.

Transforming growth factor-beta activity in sheep lung lymph during the development of pulmonary hypertension.

Chronic pulmonary hypertension is associated with extensive structural remodeling of the pulmonary arterial bed. The structural changes in the arterial walls include increased production of extracellular matrix components and smooth muscle cell hypertrophy, changes that have been similarly induced by transforming growth factor-beta (TGF-beta) in culture. In the present study, experiments were performed to determine whether TGF-beta is present in sheep lung lymph, and whether TGF-beta levels were altered in an animal model of chronic pulmonary hypertension induced by continuous air embolization. Several standard biological assays for TGF-beta activity were used for these determinations including soft agar assays, inhibition of epithelial cell proliferation, and a TGF-beta-specific radioreceptor assay. In each case, control lung lymph contained high concentrations of TGF-beta (100 ng/ml) which required transient acidification for detection. Samples of lung lymph from hypertensive sheep showed a transient and early two- to threefold increase in concentrations of latent TGF-beta. This activity could be partially blocked by TGF-beta antibodies. These studies indicate that sheep lung lymph contains TGF-beta and that the level of TGF-beta increases early during the development of pulmonary hypertension. Thus, TGF-beta may contribute to the development of the structural changes in the pulmonary arteries that occur during the onset of chronic pulmonary hypertension.

Adenoviral vector-mediated expression of physiologic levels of human factor VIII in nonhuman primates.

An E1-, E2a-, E3-deleted adenoviral vector (Av3H82) encoding an epitope-tagged B domain-deleted human factor VIII cDNA (flagged FVIII) was evaluated in nonhuman primates. Twelve cynomolgus monkeys received intravenous administration of Av3H82; 6 monkeys received 6 x 10(11) particles/kg and another 6 received 3 x 10(12) particles/kg. Adenoviral vector transduction of the liver was efficient, reproducible, and linearly dose dependent. Physiologic levels of flagged FVIII were readily detected in plasma samples obtained from monkeys that received the higher dose of vector and human FVIII mRNA was detected in their livers. Expression of transgene mRNA was restricted to the liver by the albumin promoter. Although vector DNA was readily detected in the liver of monkeys that received the lower dose, neither human FVIII mRNA nor flagged FVIII protein could be detected. Vector distribution was widespread, with the highest levels observed in liver and spleen. Histopathology, hematology, and serum chemistry analysis identified the liver and blood as major sites of toxicity. Transient mild serum elevations of liver enzymes were observed, along with a dose-dependent inflammatory response in the liver. In addition, mild lymphoid hyperplasia was observed in the spleen. Mild anemia and a transient decrease in platelet count were observed, as was marrow hyperplasia and extramedullary hematopoiesis.

Transforming Growth Factor β in the Control of Epidermal Proliferation

Transforming growth factor beta is a polypeptide growth factor with a multiplicity of diverse biologic effects. Increasingly, data support a role for TGF beta in the autocrine regulation of normal epithelial cell growth (Figure 1). Definition of the normal pathways for growth stimulation and inhibition of epithelial cell growth by autocrine peptides like TGF beta and TGF alpha undoubtedly will increase understanding of normal growth and development, embryogenesis, wound repair, and tumorigenesis.

Evaluation of the Duration of Human Factor VIII Expression in Nonhuman Primates After Systemic Delivery of an Adenoviral Vector

An E1/E2a/E3-deficient adenoviral vector encoding an epitope-tagged (flagged) human factor VIII (FVIII) cDNA was delivered systemically to four cynomolgus monkeys. Analysis of liver biopsy samples revealed the presence of vector DNA at all points in the study (day 7, 28, and 56), with vector copy number declining approximately 10-fold between day 7 and day 56. Immunoprecipitation/Western analyses detected human flagged FVIII in the plasma of all monkeys and expression persisted for 14-28 days. Peak plasma FVIII levels ranged from 50 to 100 ng/ml. Bethesda assays revealed no inhibitor in two animals, the development of a low-level transient inhibitor in one animal, and an inhibitor titer that continued to increase for the duration of the study in one animal. Other treatment-related changes included modest increases in liver enzymes, an increase in interleukin-6 (IL-6) levels, and a transient decrease in platelets in all four animals. These data indicate that early generation adenoviral vectors do not support the long-term expression of FVIII in nonhuman primates.

Transforming growth factor type β in normal human urine

Summary TGFβ has been identified in normal human urine specimens from five individuals studied for five consecutive days. The peptide was extracted from urine using Sepralyte C 1 beads. Detectable levels of [ 125 i]TGFβ competing activity as measured by radioreceptor assay was found in about half of the specimens studied. The protein isolated from urine using C 1 Sepralyte beads was further purified using Biogel P-60 column chromatography. Fractions were tested for TGFβ and EGF competing activity using radioreceptor assays. TGFβ and EGF extracted from urine are clearly separated by column chromatography. Two distinct EGF peaks and a single TGFβ peak were observed. Fractions having [ 125 1]TGFβ competing activity were pooled and further purified using reversephase HPLC. HPLC fractions having [ 125 i]TGFβ competing activity were tested for bioactivity using a soft agar assay. The fractions were capable of stimulating soft agar growth of AKR-2B (clone 84A) cells and cross reacted with a TGFβ antibody in a radioimmunoassay. The presence of TGFβ in normal human urine was also demonstrated by immunoblotting. These results also suggest that C bead extraction of urine specimens can be used as a rapid first step in purification of TGFβ.

Biological effects of transforming growth factors

Transforming growth factors (TGFs) were originally defined by their biological effects on fibroblastic cells (for review see Goustin et al. 1986). These effects included induction of morphological transformation in monolayer culture and Stimulation of colony formation in soft agar. While the early studies with TGFα were somewhat misleading with respect to the function of these factors, they did lead to the purification and cloning of two important growth-regulatory molecules, TGFα and TGFβ. Interestingly, one of these factors (TGFα) is a potent mitogen for a wide variety of cell types, while the other (TGFβ) is the most potent growth-inhibitory Polypeptide known for most cell types (Goustin et al. 1986).