Biomedical subjects

W Li

Publications and source records attributed to W Li.

At least 685 records · Page 38Linked to original sources

PubMed1998

[Construction of a competitor for bcr-abl cDNA by recombinant PCR].

OBJECTIVE: Competitive PCR is a powerful method for quantification of mRNA. This study sought to construct an internal standard which can be used as a competitor for bcr-abl fusion cDNA. METHODS: Using recombinant PCR for site-directed mutagenesis. RESULTS A 240bp mimic of a 276bp fragment in b3a2 type of bcr-abl cDNA was obtained. The difference in mimic,which was verified by DNA sequencing, was a 55bp deletion of the target sequence and a 19bp insertion of exogenous unrelated sequence with a XbaI restriction site. In it, except for the inner 36bp, the mimic contained the same sequence and shared the same primer recognition sites as the target b3a2 cDNA(276bp) or as the target b2a2 cDNA(201bp). The obtained recombinant vector, named pGEM-mimic, can be used as a common competitor for either b3a2 or b2a2 type of bcr-abl cDNA. CONCLUSION: This provided a simple and reliable method for constructing an internal standard used in competitive PCR.

DNA, Complementary↗

PubMed1998

In vivo inflammatory response to a prototypic B cell superantigen: elicitation of an Arthus reaction by staphylococcal protein A.

Staphylococcal protein A (SpA) is representative of a new class of Ags, the B cell superantigens (SAgs). These SAgs, unlike conventional Ags, bind to the Fab regions of Ig molecules outside their complementarity-determining regions. In addition, B cell SAgs can react with a substantial amount of a host's serum Igs by virtue of their ability to interact with many members of an entire variable heavy chain (VH) or variable light chain gene family. For example, SpA reacts with the Fabs of most human Igs using heavy chains from the VH3 gene family (VH3+). Members of this gene family are expressed on 30 to 60% of human peripheral B cells. We sought to determine whether the interaction of a B cell SAg with its reactive Igs can elicit immune complex-mediated tissue injury. Using the Arthus reaction in rabbits as an in vivo model of immune complex-mediated tissue inflammation, we demonstrated that untreated rabbits, which were administered SpA intradermally (i.d.), do not develop a cutaneous inflammatory response. However, when rabbits were pretreated i.v. with human IgG (hIgG), i.d. injections of SpA induced an inflammatory response with the classical histologic features of an Arthus reaction. To determine whether this Arthus-like response occurred via a B cell superantigenic mechanism, the rabbits were pretreated with VH3-depleted hIgG and then were administered SpA i.d. We found that the induction of a prominent inflammatory response by SpA was dependent upon the presence of VH3+ molecules in the hIgG pretreatment. These results provide compelling evidence that an interaction of the B cell SAg, SpA, with its reactive (VH3+) IgGs leads to an immune complex-mediated inflammatory response in vivo.

Animals↗

PubMed1998

Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1.

The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.

Binding Sites↗

PubMed1998

Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression.

Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores and triggers complex waves and oscillations in levels of cytosolic free calcium. To determine which longer-term responses are controlled by oscillations in InsP3 and cytosolic free calcium, it would be useful to deliver exogenous InsP3, under spatial and temporal control, into populations of unpermeabilized cells. Here we report the 15-step synthesis of a membrane-permeant, caged InsP3 derivative from myo-inositol This derivative diffused into intact cells and was hydrolysed to produce a caged, metabolically stable InsP3 derivative. This latter derivative accumulated in the cytosol at concentrations of hundreds of micromolar, without activating the InsP3 receptor. Ultraviolet illumination uncaged an InsP3 analogue nearly as potent as real InsP3, and generated spikes of cytosolic free calcium, and stimulated gene expression via the nuclear factor of activated T cells. The same total amount of InsP3 analogue elicited much more gene expression when released by repetitive flashes at 1-minute intervals than when released at 0.5- or > or = 2-minute intervals, as a single pulse, or as a slow sustained plateau. Thus, oscillations in cytosolic free calcium levels at roughly physiological rates maximize gene expression for a given amount of InsP3.

Animals↗

PubMed1998

Fourier transform infrared (FT-IR) spectroscopic studies of peptide models for interaction of the binding regions of high molecular weight kininogen and prekallikrein.

The binding sites for high molecular weight kininogen (HK) on prekallikrein (PK) are composed of two discontinuous segments in the primary sequence, one in Apple 1 domain (PK56=F56-G86) and the other in Apple 4 (PK266=K266-G295). The site on HK, HK31, is subsumed in a 31-amino-acid sequence (S565-K595) near the C-terminus which has the same affinity for prekallikrein as the entire HK molecule. The binding among them is likely due to conformational changes which serve to juxtapose the PK binding domain within HK with the HK binding site. Resolution-enhanced Fourier transform infrared spectroscopy (FT-IR) has been employed to analyze the contents of secondary structural elements of PK56 and HK31 and to reveal the possible specific binding portion and structural changes in HK31 and PK56 upon binding. From the amide I bands of their deconvoluted FT-IR spectra, it is known that PK56 contains no helix component, while HK31 has two different helical conformations. A quantitative comparison of the spectra of HK31, PK56 and their binding complex suggests that the conformation of 3(10)-helix in HK31 has been changed to an alpha-helix, and one disordered segment of PK56 may have been changed to extended conformation. The other structural components in PK56 and HK31 remain unchanged. Since previous studies have shown that these peptides mimic the natural protein in their bioactivity, their interaction may reflect similar changes in the natural molecules.

Amino Acid Sequence↗

PubMed1998

Aberrant regulation of transforming growth factor-alpha during the establishment of growth arrest and quiescence of growth factor independent cells.

Autocrine transforming growth factor alpha (TGFalpha) is an important positive growth effector in malignant cells and plays a significant role in generating the growth factor-independent phenotype associated with malignant progression. However, the molecular mechanisms by which TGFalpha confers a growth advantage in progression is poorly understood. The highly tumorigenic cell line HCT116 up-regulates TGFalpha mRNA expression during growth arrest, whereas the poorly tumorigenic growth factor-dependent FET cell line down-regulates TGFalpha mRNA expression as it becomes quiescent. We have identified a 25-bp sequence at -201 to -225 within the TGFalpha promoter which mediates the differential regulation of TGFalpha expression during quiescence establishment in these two cell lines. This same sequence confers TGFalpha promoter responsiveness to exogenous growth factor or autocrine TGFalpha. The abberant upregulation of TGFalpha mRNA in quiescent HCT116 cells may allow them to return to the dividing state under more stringent conditions (nutrient replenishment alone) then quiescent FET cells (requires nutrients and growth factors). Antisense TGFalpha approaches showed that the dysregulated TGFalpha expression in quiescent HCT116 cells is a function of the strong TGFalpha autocrine loop (not inhibited by blocking antibodies) in these cells.

Base Sequence↗

PubMed1998

Alpha-tocopherol prevents ethanol-induced elevation of [Ca2+]i in cultured canine cerebral vascular smooth muscle cells.

Exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10-400 mM) for 1-5 days results in concentration-dependent elevation in resting intracellular free calcium ([Ca2+]i) levels. Preincubation of these cultured vascular cells with alpha-tocopherol (20 microM), alone, did not produce any apparent changes from control resting levels of [Ca2+]i. However, after concomitant addition of alpha-tocopherol (20 microM) and ethanol (10-400 mM), the rises of [Ca2+]i induced by ethanol were attenuated markedly. These results suggest that alcohol-induced lipid peroxidation of cerebral vascular muscle cell membranes triggers membrane entry of extracellular Ca2+, which could play an important role in ethanol-induced cerebrovasospasm, brain ischemia and stroke. Moreover, these new results support the concept recently advanced to suggest that alpha-tocopherol-induced amelioration of membrane lipid alterations of cerebral vascular cells can prevent ethanol-induced excessive accumulation of [Ca2+]i.

Animals↗

PubMed1998

Regulation of transforming growth factor-beta type II receptor expression in human breast cancer MCF-7 cells by vitamin D3 and its analogues.

In view of the tumor suppressor role of the transforming growth factor-beta (TGFbeta) type II receptor (RII), the identification and characterization of agents that can induce the expression of this receptor are of potential importance to the development of chemoprevention approaches as well as treatment of cancer. To date, the identification of exogenous agents that control RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGFbeta growth inhibition activity, was significantly inhibited by vitamin D3 and its analogue EB1089. In contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which have lost cell surface RII and are resistant to TGFbeta, was not affected by these two compounds. TGFbeta-neutralizing antibody was able to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGFbeta activity. An RNase protection assay showed approximately a 3-fold induction of the RII mRNA, while a receptor cross-linking assay revealed a 3-4-fold induction of the RII protein. In contrast, there was no change in either RII mRNA or protein in the MCF-7 L cells.

Antineoplastic Agents↗

PubMed1998

Regulatory effect of CD9 on calcium-stimulated phosphatidylserine exposure in Jurkat T lymphocytes.

Alteration in membrane-phosphatidylserine (PS) asymmetry occurs during lymphocyte apoptosis, but the mechanism and regulation of this process are not well understood. We investigated the possible involvement of CD9, a member of the tetraspan family of membrane proteins, in the PS-exposure process in Jurkat T cells; flow cytometry with FITC-annexin V was used to detect the PS-positive cells. We found that antibody to CD9 inhibited the prompt (within 5 min) PS exposure stimulated by calcium ionophore A23187, whereas it had no effect on resting cells. Antibodies against other control antigens (CD7, CD56, CD57, CD59, CD63, and CD71) had no effect on PS exposure in either resting or calcium-ionophore-treated cells. The inhibitory effect of anti-CD9 was dose dependent. The observed inhibitory effect appeared to be "all or none" at the cellular level: increasing antibody doses decreased the percentage of PS-positive cells, whereas the number of PS molecules exposed per positive cell was constant. The inhibitory effect was not blocked by co-incubation with other antibodies of the same isotype, arguing against a nonspecific effect via Fc receptors. The anti-CD9, however, did not block the delayed (8- to 24-h) PS exposure induced by apoptotic treatments such as ultraviolet light, cycloheximide, and actinomycin D, indicating that CD9 might act selectively on only some pathways leading to PS exposure. Our results suggest that lymphocyte PS exposure is regulated by multiple pathways and that signals regulating PS exposure can be delivered through cell-surface antigen CD9.

Annexin A5↗

PubMed1998

Donor bone marrow potentiates the effect of tacrolimus on nonvascularized heart allograft survival: association with microchimerism and growth of donor dendritic cell progenitors from recipient bone marrow.

BACKGROUND: The influence of donor hematopoietic cell microchimerism on organ allograft survival has been studied largely in vascularized transplant models. Here, we examine the impact of donor bone marrow (BM) cells administered intravenously together with transient systemic tacrolimus therapy on microchimerism, the survival of nonvascularized cardiac allografts, and growth of donor antigen-presenting cells [dendritic cells (DCs)] from recipient BM. METHODS: Adult male C3H (H2k) mice received heterotopic heart transplants from B10 (H2b) donors in the dorsal ear pinna. They were given no further treatment, or either a short course of tacrolimus (FK506; 2 mg/kg i.p. from day 0 to day 13), unmodified donor BM cells (50x10(6) i.v. on day 0) or both treatments. Grafts were examined daily for contractile activity. Anti-donor cytotoxic T lymphocyte responses were determined in recipients' spleens. Microchimerism (IAb+ cells) was demonstrated by immunocytochemical staining of spleens, and of cells expanded from recipient BM using cytokines and culture conditions that promote the growth of DCs. RESULTS: Tacrolimus alone significantly prolonged median heart graft survival time from 10 to 22 days (P<0.001). BM alone failed to prolong graft survival. By contrast, tacrolimus + donor BM resulted in a mean survival time of 42 days (P<0.01 compared with tacrolimus treatment alone). This marked increase in heart allograft survival was associated with reduced anti-donor cytotoxic T lymphocyte responses attributable to a nonspecific effect of tacrolimus. In addition, however, a link was observed between the beneficial effect of donor BM and comparatively large numbers of donor major histocompatibility complex class II (IAb+)-positive cells in recipients' spleens, and in cultures of granulocyte-macrophage colony-stimulating factor + interleukin-4-stimulated DCs from recipients' BM. No donor-derived cells were propagated from heart graft recipients given either tacrolimus or donor BM alone. CONCLUSIONS: This nonvascularized organ transplant model demonstrates the positive effect on allograft survival of donor BM given at the time of transplant to transiently immunosuppressed recipients. The findings also reveal links between hematopoietic cell chimerism, the presence of donor DC progenitors in recipient BM, and organ allograft survival.

Animals↗

PubMed1998

Prolonged depletion of AH receptor without alteration of receptor mRNA levels after treatment of cells in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Previous experiments have shown that the total cellular content of the AH receptor (AHR) drops rapidly after exposure of mouse hepatoma cells (Hepa-1) to the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); within 6 hr after treatment, less than 20% of the original cell content of AHR can be detected by radioligand binding or by immunoblotting. The goals of our current study were to determine the duration of receptor depletion following treatment with ligand and to determine if depletion is due to decreased expression of the Ahr gene that encodes the AHR. We found that depletion of AHR persisted for at least 72 hr after exposure to TCDD. Treatment with 3-methylcholanthrene caused a transient drop in total cell AHR, but the AHR levels returned to near pretreatment levels within 72 hr after the first exposure. TCDD treatment did not alter the levels of AHR mRNA as assessed by reverse transcription-polymerase chain reaction or slot blot assays. Thus, the decrease in AHR protein cannot be attributed to depression of transcription of the Ahr gene by TCDD. TCDD treatment did not alter the levels of the dimerization partner of the AHR, the AH receptor nuclear translocator protein (ARNT), or ARNT mRNA. In the presence of TCDD, both the AHR and the ARNT protein can be maintained at high levels in the nucleus if transcription is inhibited with actinomycin-D. In the absence of actinomycin-D, the AHR protein was lost rapidly, but the ARNT protein level in the cell was maintained. Together, these results suggest that the AHR protein is degraded through a selective mechanism that spares the ARNT protein and that the degradation pathway involves a protein that itself has a short half-life.

Animals↗

PubMed1998

Mammalian DNA topoisomerase IIIalpha is essential in early embryogenesis.

Targeted disruption of the mouse TOP3alpha gene encoding DNA topoisomerase IIIalpha was carried out to study the physiological functions of the mammalian type IA DNA topoisomerase. Whereas heterozygous top3alpha+/- mutant mice were found to resemble phenotypically their TOP3alpha+/+ litermates, no viable top3alpha-/- homozygotes were found among over 100 progeny of top3alpha+/- intercrosses. Examination of embryos dissected from decidual swellings and in vitro culturing of blastocysts from top3alpha+/- intercrosses showed that implantation of top3alpha-/- embryos and the induction of decidualization could occur, but viability of these embryos was severely compromised at an early stage of development. The requirement of mouse DNA topoisomerase IIIalpha during early embryogenesis is discussed in terms of its plausible role in chromosome replication and its interaction with the RecQ/SGS1 family of DNA helicases, whose members include the Bloom's syndrome and the Werner's syndrome gene products.

Animals↗

PubMed1998

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate.

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins.

3T3 Cells↗

PubMed1998

Staurosporine and H7 attenuate ethanol-induced elevation in [Ca2+]i in cultured canine cerebral vascular smooth muscle cells.

Chronic exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10-400 mM) for 1-5 days resulted in significant concentration-dependent elevation in resting intracellular free calcium ([Ca2+]i) levels. Preincubation of these cultured vascular cells with inhibitors of protein kinase C (PKC), staurosporine and H7, induced no apparent changes from the control resting levels of [Ca2+]i. However, the increases of [Ca2+]i due to ethanol treatment were attenuated markedly by staurosporine and H7. Our data suggest that activation of PKC plays an important role in ethanol's action in producing a sustained rise in [Ca2+]i in cerebral vascular smooth muscle cells. Activation of PKC could thus play a crucial role in the pathogenesis of alcohol-induced cerebral ischemia and stroke.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Retinopathy in older persons without diabetes and its relationship to hypertension.

OBJECTIVE: To assess the prevalence and relationship of retinopathy lesions in older subjects without diabetes to systemic hypertension. METHODS: Three thousand six hundred fifty-four people aged 49 years or older attending the Blue Mountains Eye Study underwent a detailed eye examination, including medical history, blood pressure measurement, and fasting blood collection. Retinopathy lesions (hemorrhages and microaneurysms) were assessed from masked grading of stereoretinal photographs. Subjects with a history of diabetes or an elevated blood glucose level were excluded. Hypertension was defined as the current use of antihypertensive medications or an elevated blood pressure measurement on examination. RESULTS: Retinopathy was present in 325 subjects without diabetes, a prevalence of 9.8% (95% confidence interval [CI], 8.9%-10.9%), which increased with age. An increased age-adjusted relative risk (RR) for retinopathy was found for women (RR, 1.67; 95% CI, 1.26-2.21) and men (RR, 1.47; 95% CI, 1.07-2.00) with hypertension. In people using antihypertensive medications, retinopathy prevalence was higher for uncontrolled compared with controlled blood pressure but was not related to hypertension duration. Significant (P=.02 to P=.001) trends were found between increasing blood pressure quartiles and age-adjusted retinopathy prevalence, a relationship that was maintained after adjusting for fasting plasma glucose level at 3 diagnostic cut points for diabetes. CONCLUSIONS: This study supports the Beaver Dam Eye Study findings that retinal hemorrhages and microaneurysms are relatively frequent lesions in older people without diabetes and are significantly related to the presence and severity of hypertension.

Aged↗

PubMed1998

ECG-gated versus nongated three-dimensional intracoronary ultrasound analysis: implications for volumetric measurements.

The quantitative analysis of a three-dimensional (3-D) intracoronary ultrasound (ICUS) image data set permits a more comprehensive assessment of coronary arterial segments. The 3-D image sets are generally acquired during continuous motorized pullbacks. However, the cyclic changes of vascular dimensions and the cyclic spatial displacement of the ICUS transducer relative to the vessel wall can result in characteristic image artifacts, which may limit the applicability of quantitative automated analysis systems. This limitation may be overcome by an ECG-gated image acquisition. In the present study we acquired in vivo (1) nongated and (2) ECG-gated 3-D ICUS image sets of 15 human atherosclerotic coronary arteries and performed a computer-assisted contour detection of the lumen and total vessel boundaries. Total vessel and lumen volumes measured significantly larger in the nongated versus ECG-gated end-diastolic image sets (753+/-307 mm3 vs. 705+/-305 mm3; 411+/-154 mm3 vs. 388+/-165 mm3, both: P < 0.05). Both end-diastolic and systolic measurements were available in nine arteries, showing a larger total vessel and lumen volume at systole (664+/-221 mm3 vs. 686+/-227 mm3, P=0.03; 384+/-164 mm3 vs. 393+/-170 mm3, P=0.08). The differences observed may be of particular interest for volumetric ICUS studies, addressing presumably small differences in vessel or lumen dimensions.

Adult↗

PubMed1998

Expression of transforming growth factor-beta receptor type II and tumorigenicity in human breast adenocarcinoma MCF-7 cells.

To analyze transforming growth factor-beta (TGF-beta) response during MCF-7 cell progression, early passage (MCF-7E, < 200 passage) and late passage (MCF-7L, > 500 passage) cells were compared. MCF-7E cells showed an IC50 of approximately 10 ng/ml of TGF-beta1, whereas MCF-7L cells were insensitive. MCF-7E cells contained approximately threefold higher levels of TGF-beta receptor type II (TbetaRII) mRNA than MCF-7L, but their TbetaRI levels were similar. MCF-7E parental cells showed higher TbetaRII promoter activity than MCF-7L cells, which could be attributed to changes in Sp1 nuclear protein levels. Receptor cross-linking studies indicated that the cell surface receptor levels parallel mRNA levels in both cell lines. Limiting dilution clones of MCF-7E cells were established to determine the heterogeneity of TbetaRII expression in this cell line, and they showed varying degrees of TbetaRII expression. Fibronectin was induced at higher levels in cells expressing higher TbetaRII levels. All three TGF-beta isoforms were detected in limiting dilution clones and parental cells, but TGF-beta1 was more abundant relative to TGF-beta2 or 3, and no correlation between TGF-beta isoform profile with TGF-beta sensitivity was found. MCF-7L cells were tumorigenic and formed xenografts rapidly and progressively, whereas MCF-7E parental and limiting dilution clonal cells showed transient tumor formation followed by regression. These results indicate that decreased TbetaRII transcription in breast cancer cells leads to a loss of TbetaRII expression, resulting in cellular resistance to TGF-beta which contributes to escape from negative growth regulation and tumor progression.

Adenocarcinoma↗

PubMed1998

Autocrine TGFalpha expression in the regulation of initiation of human colon carcinoma growth.

Previously, we reported that unaggressive, growth factor-dependent FET human colon carcinoma cells downregulated their transforming growth factor alpha (TGFalpha) expression in a quiescent state (G0/G1) induced by growth factor and nutrient deprivation (Mulder, 1991, Cancer Res., 51:2256-2262). In contrast, highly aggressive, growth factor-independent HCT116 human colon carcinoma cells aberrantly upregulated this autocrine activity in the quiescent state (Mulder, 1991, Cancer Res., 51:2256-2262; Howell et al., 1998, Mol. Cell. Biol., 18:303-313). In this report, the role of autocrine TGFalpha and the mechanism of its regulation of expression during reentry into the cell cycle from a noncycling growth state were determined in FET cells. Optimal induction of DNA synthesis from a quiescent state in FET cells is dependent upon autocrine TGFalpha as well as exogenous transferrin and insulin. Reentry into the cell cycle resulting from treatment with exogenous transferrin and insulin resulted in approximately 3-fold induction of TGFalpha expression within 1 hr. TGFalpha induction was controlled at the transcription level, and the cis-controlling element was localized to the region between bp -370 - -201 relative to the translation start codon within the TGFalpha promoter. Thus neutralization of autocrine TGFalpha protein revealed that the induced TGFalpha autocrine activity was necessary for DNA synthesis and acted only in the early G1 phase of the cell cycle. Blockade of autocrine TGFalpha expression early in the cell cycle resulted in the reduction of DNA synthesis, whereas treatment with neutralization antibody at later times had no effect. This suggested that autocrine TGFalpha functions to initiate cell growth from noncycling states. This was further confirmed by the dependence of FET cells upon autocrine TGFalpha for colony formation in experiments where the plating density was sufficiently low to generate a lag phase in tissue culture. In contrast, TGFalpha autocrine activity was not required for exponential phase cells, as evidenced by the failure of TGFalpha neutralizing antibody to inhibit proliferation in this growth state. Taken together, these results suggest that autocrine TGFalpha acts primarily in the process of growth initiation by moving cells from a noncycling state back into the cell cycle, rather than supporting cell growth already initiated.

Autocrine Communication↗