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PubMed2003

Competing autocrine pathways involving alternative neuropilin-1 ligands regulate chemotaxis of carcinoma cells.

Neuropilin-1 (NP1), in conjunction with plexins, promotes axon repulsion by binding to semaphorin 3A (SEMA3A). Although NP1 is expressed in carcinoma cells, its functions have remained elusive, and neither SEMA3A nor plexin expression has been explored in cancer. Here we provide evidence that breast carcinoma cells support an autocrine pathway involving SEMA3A, plexin-A1, and NP1 that impedes their ability to chemotax. Reducing SEMA3A or NP1 expression by RNA interference or inhibiting plexin-A1 signaling enhanced migration. Conversely, expression of constitutively active plexin-A1 impaired chemotaxis. The paradox of how breast carcinoma cells expressing these endogenous chemotaxis inhibitors are able to migrate is explained by their expression of vascular endothelial growth factor (VEGF), a NP1 ligand that competes with SEMA3A for receptor binding. Finally, we establish that the ratio of endogenous VEGF and SEMA3A concentrations in carcinoma cells determines their chemotactic rate. Our findings lead to the surprising conclusion that opposing autocrine loops involving NP1 regulate the chemotaxis of breast carcinoma cells. Moreover, our data indicate a novel autocrine function for VEGF in chemotaxis.

Breast Neoplasms↗

PubMed2003

Epidermal growth factor receptor autocrine signaling in RIE-1 cells transformed by the Ras oncogene enhances radiation resistance.

Oncogenic forms of the small GTPase Ras increase the resistance of cells to killing by ionizing radiation (IR). Although not all of the signaling pathways for radioresistance are well defined, it is now clear that Ras-dependent signaling pathways involved in radioresistance include those mediated by phosphatidylinositol 3'-kinase (PI3-K) and Raf. Nevertheless, PI3-K and Raf together are not sufficient to reconstitute all of the resistance conferred by Ras, indicating that other effectors must also contribute. We show here that Ras-driven autocrine signaling through the epidermal growth factor receptor (EGFR) also contributes to radioresistance in Ras-transformed cells. Conditioned media (CM) collected from RIE-1 rat intestinal epithelial cells expressing oncogenic Ras increased the survival of irradiated cells. Ras-CM contains elevated levels of the EGFR ligand transforming growth factor alpha (TGF-alpha). Both Ras-CM and TGF-alpha stimulated EGFR phosphorylation, and exogenous TGF-alpha mimicked the effects of Ras-CM to increase radioresistance. Blocking EGFR signaling with the EGFR/HER-2 kinase inhibitor (KI) GW572016 decreased the postradiation survival of irradiated Ras-transformed cells and normal cells but had no effect on the survival of unirradiated cells. Ras-CM and TGF-alpha also increase PI3-K activity downstream of the EGFR and increase postradiation survival, both of which are abrogated by GW572016. Thus, Ras utilizes autocrine signaling through EGFR to increase radioresistance, and the EGFR KI GW572016 acts as a radiosensitizer. The observation that Ras-transformed cells can be sensitized to killing by ionizing radiation with GW572016 demonstrates that EGFR KIs could potentially be used to radiosensitize tumors in which radioresistance is dependent on Ras-driven autocrine signaling through EGFR.

Animals↗

PubMed1991

Evidence for the involvement of transforming growth factor alpha and epidermal growth factor receptor autocrine growth mechanism in primary human ovarian cancers in vitro.

We examined 35 primary human ovarian adenocarcinomas for the presence of epidermal growth factor (EGF) receptor (EGFR) in plasma membranes from cancer tissues by using 125I-EGF as a ligand. Specific 125I-EGF bindings were observed in 20 (57%) of these 35 cases. Scatchard analysis showed a class of high affinity EGF receptor: Kd 5.0 +/- 1.0 x 10(-10) M; Bmax, 83.3 +/- 12.1 fmol/mg protein (mean +/- SE, n = 20). Northern analysis in polyadenylated RNA from 15 EGFR(+) cancers using pretransforming growth factor alpha (pre-TGF alpha), prepro-EGF complementary DNA, and pE7, a complementary DNA clone of human EGFR, as probes revealed that pre-TGF alpha and EGFR mRNAs but not prepro-EGF mRNA were expressed in all cases examined. Immunocytochemical studies using monoclonal antibodies (mAbs) against TGF alpha, EGF, and EGFR showed that TGF alpha and EGFR but not EGF proteins were present on ovarian cancer cells in all cases. These data suggested a possible TGF alpha/EGFR autocrine mechanism in EGFR(+) ovarian cancers. We, therefore, examined the biological significance of this autocrine mechanism by using primary monolayer cell cultures. In primary cultures from EGFR (+) cancers, TGF alpha added to the culture medium stimulated the [3H]thymidine incorporation dose dependently. Moreover, the addition of mAbs against TGF alpha and EGFR but not EGF inhibited [3H]thymidine incorporation dose dependently in EGFR(+) cancer cells. On the other hand, in primary cultures from EGFR(-) cancers, TGF alpha and anti-TGF alpha, -EGFR, and -EGF mAbs did not show any effects on [3H]thymidine incorporation. All these results suggested the possible crucial role of a TGF alpha/EGFR autocrine growth mechanism in primary human ovarian cancers which express EGFR.

Adenocarcinoma↗

PubMed1991

Involvement of transforming growth factor alpha/epidermal growth factor receptor autocrine growth mechanism in an ovarian cancer cell line in vitro.

Although transforming growth factor (TGF) alpha and epidermal growth factor (EGF) receptor (EGFR) autocrine mechanism is widely demonstrated in many kinds of cancers, its biological significances still remain circumstantial. We critically assessed the significance of this mechanism on the growth of an ovarian cancer cell line. Northern blot analysis in polyadenylated RNA isolated from cells by using 32P-labeled pre-TGF alpha, EGRF, and prepro-EGF complementary DNAs as probes revealed that pre-TGF alpha and EGFR but not prepro-EGF gene transcripts were expressed in the cell. TGF alpha and EGFR but not EGF proteins were observed by immunocytochemical stainings, using monoclonal antibodies against human TGF alpha, EGFR, and EGF, respectively. This cell line possessed a class of high affinity EGF receptor by 125I-EGF binding studies; Kd being 2.9 x 10(-10) M and Bmax to be 7.7 x 10(4) sites/cell. As much as 1.12 +/- 0.14 ng (SD; n = 3)/10(7) cells/24 h of TGF alpha was secreted in the conditioned media. These results suggested the expression of a TGF alpha/EGFR autocrine mechanism in this cell line. We, therefore, assessed the biological significance of this mechanism on the growth of this cell line in serum-free monolayer cell cultures. Although 0.1, 1.0, and 10 nM concentrations of TGF alpha did not show significant growth promotion, monoclonal antibodies against TGF alpha and EGFR but not EGF significantly inhibited cell growth. All these data suggested the biological importance of a TGF alpha/EGFR autocrine mechanism on the growth of this cell line in vitro.

Antibodies, Monoclonal↗

PubMed1991

Autocrine activity of interleukin 6 secreted by hepatocarcinoma cell lines.

Among several rat hepatoma cell lines known to secrete interleukin 6 (IL6), the HTC.JZ1 line stands out as a high-level producer. HTC.JZ1 cells were stimulated to secrete up to fourfold increased amounts of IL6 over 24 hours by treatment with lipopolysaccharides (LPS). Both functional IL6 levels, measured as hepatocyte stimulating factor (HSF) activity, and IL6 mRNA concentrations were increased proportionally by exposure to LPS. Similarly, IL6 mRNA was induced by LPS treatment in cultured primary rat hepatocytes. The induction of Il6 mRNA by LPS was inhibited both in primary hepatocyte and hepatoma cell cultures by treatment with the synthetic glucocorticoid dexamethasone, consistent with the known analogous repression of the IL6 gene by dexamethasone in macrophages, monocytes and fibroblasts. IL6 secreted by HTC.JZ1 cells was utilized as an autocrine inducer of endogenous acute phase gene expression: HTC cells expressed constitutive levels of alpha 2-macroglobulin (alpha 2M) mRNA specified by the major rat acute phase gene, the alpha 2M gene, which is known to be regulated by IL6. By contrast, normal rat liver biopsy material and a number of other rat hepatoma cell lines lacked endogenous IL6 production and showed very low to zero expression of endogenous alpha 2M mRNA. Expression of alpha 2M mRNA in HTC.JZ1 cells was inducible by treatment with LPS. The constitutive and the LPS-induced production of alpha 2M mRNA were significantly reduced (up to 50% inhibition) by addition of an anti IL6 serum to the culture medium and removal of the immune complexes. However, complete neutralization of the alpha 2M-inducing HSF activity could not be obtained with anti-IL6 serum alone, probably because HTC.JZ1 cells secrete comparable quantities of a second HSF activity. This activity, the cytokine leukemia inhibitory factor (LIF), is also known to stimulate transcription of the rat alpha 2M gene but was not reactive with anti-IL6 sera. The induction of IL6 mRNA in HTC cells by LPS was regulated at the transcriptional level, as demonstrated by a series of mutagenesis and transfection experiments. Progressive deletion of 5' flanking sequences from the IL6 gene promoter region reduced the basal level, and the LPS-induced promoter activity after transfection into HTC.JZ1 hepatoma cells. IL6 has been shown to act as an autocrine regulator of growth for certain B lymphoid cell lines derived from human multiple myelomas. The results presented here establish that IL6 secreted by certain hepatoma cell lines also acts in an autocrine fashion to induce expression of the endogenous acute phase alpha 2M gene.

Animals↗

PubMed1991

Importance of transforming growth factor alpha/epidermal growth factor receptor autocrine growth mechanism in an ovarian cancer cell line in vivo.

We have elucidated the importance of a transforming growth factor (TGF) alpha and epidermal growth factor receptor autocrine mechanism on the growth of a human ovarian serous cystadenocarcinoma-derived cell line (SHIN-3) in vitro. In this study, we studied the biological significance of this autocrine mechanism in vivo using female athymic nude (nu/nu) mice. We measured the mouse plasma epidermal growth factor and TGF alpha levels by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Plasma epidermal growth factor concentrations were remarkably decreased by sialoadenectomy (Sx): 410 +/- 65 (SE) pg/ml (n = 10) in intact animals; and undetectable in Sx mice (n = 5). Plasma TGF alpha levels were 90 and 40 pg/ml in intact and in Sx animals, respectively. Ten million SHIN-3 cells inoculated into nu/nu mice formed tumors in 100% of mice, and tumors grew progressively. Implantabilities and tumor growth rates of inoculated cells were not affected by Sx and even by Sx and anti-mouse epidermal growth factor antibody treatment. However, anti-TGF alpha monoclonal antibody (mAb) administered to SHIN-3 cell-inoculated Sx animals drastically reduced the tumor growth. Although 10(7) SHIN-3 cells formed tumors in this group, tumor growth was significantly inhibited by 10 micrograms of anti-TGF alpha mAb given 3 times a week, and growth inhibitions were more by 20 micrograms of anti-TGF alpha mAb. Moreover, as aggressive tumor growth as that in Sx animals was resumed by the cessation of anti-TGF alpha mAb treatments. All these data suggested the biological importance of a TGF alpha/epidermal growth factor receptor autocrine mechanism on the growth of this cell line in vivo.

Animals↗

PubMed1991

Activation of the autocrine transforming growth factor alpha pathway in human squamous carcinoma cells.

Transforming growth factor alpha is an autocrine mitogen for nonneoplastic keratinocytes, which exerts its function by binding to the receptor for epidermal growth factor. In order to determine whether this autocrine pathway is activated in squamous carcinoma cells, we analyzed the production of transforming growth factor alpha as well as the expression and regulation of epidermal growth factor receptors in a panel of human squamous carcinoma cell lines. Immunoreactive transforming growth factor alpha was detectable in squamous carcinoma cells as well as in quiescent nonneoplastic keratinocytes. However, in the absence of exogenous mitogens, only the squamous carcinoma cells secreted the growth factor into the medium, whereas untransformed keratinocytes did not. Each of the squamous carcinoma cell lines expressed significantly greater numbers of cell surface epidermal growth factor receptors than normal keratinocytes. The epidermal growth factor receptor gene was amplified and overexpressed in three of the squamous carcinoma cell lines (A431, CaSki, SqCC/Y1). Two of the squamous carcinoma cell lines (C4-1 and CE-48) displayed a relative inability to down-regulate epidermal growth factor receptors in response to epidermal growth factor. The mechanism of receptor overexpression in the remaining three cell lines (A253, CaLu-1, FaDu) is unexplained. Thus, human squamous carcinoma cell lines frequently exhibit a combination of the constitutive secretion of transforming growth factor alpha and the overexpression of epidermal growth factor receptors. Treatment of these tumor cells with an antibody directed against the ligand-binding domain of the epidermal growth factor receptor inhibited their growth by approximately 50%. These findings suggest that designing strategies to interrupt the transforming growth factor alpha autocrine pathway might lead to new modalities to treat this class of malignant tumors.

Carcinoma, Squamous Cell↗

PubMed1990

Epstein-Barr virus-immortalized B cells produce IL-6 as an autocrine growth factor.

The continuous proliferation of Epstein-Barr virus (EBV)-immortalized B cells is enhanced by autocrine as well as paracrine growth factors. In the present study, the possibility that EBV-immortalized B cells might produce interleukin-6 (IL-6) proteins in an autocrine manner was examined. It was found that culture supernatants from EBV-transformed B cells, but not from Burkitt's lymphoma lines, augmented the proliferation of an IL-6-dependent murine hybridoma clone, MH60.BSF2. This growth-promoting activity for hybridoma cells found in culture supernatants of EBV-transformed B cells was specifically neutralized by rabbit anti-recombinant (r) IL-6 antibody. The IL-6 activity in culture supernatants of EBV-transformed B cells, though much less than that of lipopolysaccharide (LPS)-stimulated monocytes, was increased by the addition of phorbol myristate acetate. Western blot experiments using rabbit anti-rIL-6 antiserum demonstrated that supernatants from cultured EBV-transformed B cells contained the distinct forms of IL-6, with a peak of 23,000 MW. When examined by in situ hybridization analysis, it was found that IL-6 mRNA were expressed on EBV-transformed B cells. It was noted that a fraction, but not all, of these cells expressed IL-6 mRNA strongly, implying their cell cycle-dependent expression. In addition, it was shown that rIL-6 promoted the growth of EBV-transformed B cells at low cell densities. The results suggest that IL-6 serves as an autocrine growth factor in EBV-transformed B cells.

B-Lymphocytes↗

PubMed1990

Transforming growth factor beta 1 acts as an autocrine-negative growth regulator in colon enterocytic differentiation but not in goblet cell maturation.

Previous studies from this laboratory (Schroy, P., Rifkin, J., Coffey, R.J., Winawer, S., and Friedman, E. (Cancer Res., 50: 261-265, 1990; Schroy, P.C., Winawer, S., and Friedman, E. Cancer Lett., 48: 53-58, 1989) found that a 7-day treatment of the human colon carcinoma cell line HT29 with the differentiation agent hexamethylene bisacetamide (HMBA) induces both a 4-5-fold increase in transforming growth factor beta 1 (TGF beta 1) mRNA levels and reduced tumorigenicity in vivo. A series of 15 cloned lines with different commitments to differentiation has been isolated from 20-day HMBA-treated HT29 cells, maintained without HMBA, and utilized to study the role of TGF beta 1 in colon carcinoma differentiation. Two such lines, HD6 and HD8, differentiate to 97 and 76% mucus-secreting goblet cells, respectively, in columnar monolayers in postconfluent culture. Both HD6 and HD8 cells exhibit low TGF beta 1 mRNA levels, little different from the undifferentiated HT29 parental line, and exhibit no growth modulation in response to exogenous TGF beta 1. In contrast, two other lines, HD3 and HD4, differentiate to fluid-transporting enterocytic cells with functional brush borders and exhibit autocrine-negative growth response to TGF beta 1. Both lines express TGF beta 1 mRNA at levels 11-12-fold higher than the parental line and respond to exogenous TGF beta 1 by growth inhibition. HD3 cells secrete biologically active TGF beta 1 into conditioned media, which inhibited growth of a TGF beta 1-sensitive mink cell line. This inhibition was blocked by antisera to TGF beta 1, proving the specificity of the inhibition. A range of concentrations of this TGF beta 1 antiserum stimulated HD3 cell growth in a dose-dependent manner, further documenting the autocrine-negative response of the cells to TGF beta 1. Another cell line, HI1, was blocked in enterocytic differentiation. HI1 cells synthesized as much TGF beta 1 mRNA as HD3 and HD4 cells, yet they responded to exogenous TGF beta 1 with less growth inhibition, suggesting some impairment in their response to TGF beta 1. A third class of response to TGF beta 1 was exhibited by the HP1 cell line, which was resistant to HMBA-induced differentiation, remaining undifferentiated with a multilayered growth pattern. HP1 cells synthesized TGF beta 1 mRNA at levels over 20 times the parental level but were stimulated to divide by TGF beta 1, exhibiting autocrine-positive response to this growth factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetamides↗

PubMed1989

Expression of growth factors and growth factor receptors in soft tissue tumors. Implications for the autocrine hypothesis.

According to the autocrine hypothesis of cell growth, tumors, in contrast to normal tissue, might simultaneously express both a growth factor and its receptor, resulting in autoregulation and autostimulation of growth. In a related hypothesis, expression of either a growth factor or receptor might allow a tumor to escape exogenous controls on growth, and thereby correlate with malignant potential. Using immunohistochemistry, we investigated these hypotheses in 69 human soft tissue tumors (STT) by assaying for the expression of nerve growth factor and receptor, epidermal growth factor and receptor and platelet-derived growth factor. Both benign (B) and malignant (M) STT expressed a variety of factors and receptors. However, the frequency of any growth factor expression (90% M versus 55% B, p = 0.002) and of multiple factors (59% M versus 24% B, p = 0.006) was significantly greater in malignant tumors. Similarly, expression of any growth factor receptor (73% M versus 31% B, p = 0.001) and of multiple receptors (35% M versus 3% B, p = 0.002) was significantly more frequent in malignant STT. In terms of the autocrine hypothesis, growth factor/receptor co-expression was significantly more common in malignant STT (63% M versus 17% B, p = 0.0002). We conclude that (a) expression of both single and multiple growth factors and receptors was significantly more frequent in malignant STT; (b) support for an autocrine growth mechanism through simultaneous factor/receptor co-expression can be found in both benign and malignant STT; (c) co-expression, however, was more frequent in the malignant tumors; and (d) overall, growth factor and receptor expression, as well as co-expression, was related to biologic potential among human soft tissue tumors.

Epidermal Growth Factor↗

PubMed1989

Tumor necrosis factor acts synergistically with autocrine interferon-beta and increases interferon-beta mRNA levels in human fibroblasts.

Medium of untreated human FS-4 foreskin fibroblasts contained a factor which, upon the addition of exogenous tumor necrosis factor (TNF), inhibited encephalomyocarditis virus replication when neither medium alone nor TNF alone were effective. This antiviral activity was abolished by a monoclonal antibody to human interferon (IFN)-beta, suggesting that the active component in the medium from untreated FS-4 cells was IFN-beta, present at subeffective concentrations. In addition, we show that untreated FS-4 cells contain IFN-beta mRNA, demonstrable by the highly sensitive polymerase chain reaction after reverse transcription. Treatment of FS-4 cells with TNF produced an approximately 16-fold increase in the steady-state level of IFN-beta mRNA. Our results support the conclusion that autocrine IFN-beta is secreted by untreated normal fibroblasts and that TNF can enhance the production of autocrine IFN-beta by increasing the level of IFN-beta mRNA. Our study also demonstrates that subeffective concentrations of autocrine IFN-beta, which escape detection in conventional assays, are sufficient to produce a strong synergistic action with TNF.

Antibodies, Monoclonal↗

PubMed1989

Autocrine growth factor in defined serum-free medium of human salivary gland adenocarcinoma cell line HSG.

Human salivary gland adenocarcinoma cell line HSG secretes an epidermal growth factor (EGF)-like molecule and contains EGF receptors. Growth of HSG cells is inhibited by glucocorticoid. We have identified that the growth inhibition by glucocorticoid is induced by the reduced secretion of the EGF-like molecule and that addition of anti-human EGF antibody to the culture specifically inhibits the growth of HSG cells, suggesting that autocrine secretion is involved in the growth of HSG cells. To prove that autocrine secretion functions in glucocorticoid-regulated growth of the HSG cell line, we purified the EGF-like molecule from serum-free, defined medium conditioned by the HSG cells and examined the growth-stimulatory effect of the purified molecule. The cultivation of HSG cells in serum-free defined medium, which contains insulin (10 micrograms/ml) and transferrin (10 micrograms/ml) only as proteinaceous components, resulted in establishment of a new cell line (HSG-SF) which had different morphological features from the parental HSG cell line. HSG-SF cells were found to have basically the same responsiveness to glucocorticoid as parental HSG cells. Parental HSG cells secreted high molecular weight EGF-like molecules (Mr 46,000 and 57,000), which were recognized by specific antibody to low molecular weight human EGF (Mr 6,201). From conditioned, serum-free medium of HSG-SF cells, an EGF-like molecule (Mr 46,000) was purified by using an anti-human EGF antibody-coupled Sepharose CL-4B column. This EGF-like molecule induced a maximal increase (36%) in incorporation of [3H]thymidine into DNA of parental HSG cells as well as low molecular weight human EGF. These observations demonstrate that growth of the HSG cell line is regulated by autocrine secretion.

Adenocarcinoma↗

PubMed1988

An autocrine factor from Reuber hepatoma cells that stimulates DNA synthesis and acetyl-CoA carboxylase. Characterization of biologic activity and evidence for a glycan structure.

Conditioned medium from Reuber H-35 or Fao hepatoma cells contains autocrine factors that both stimulate DNA synthesis and activate acetyl-coenzyme A (CoA) carboxylase in serum-deprived Fao cells. The factor(s), which appears within 4 h of serum-free culture, also increases the cell number and the mitotic index. The effects of the conditioned medium are insulinomimetic, both with respect to stimulation of DNA synthesis and acetyl-CoA carboxylase activity. However, no induction of tyrosine aminotransferase activity or stimulation of aminoisobutyric acid uptake is seen in response to the conditioned medium. Insulin over a 4-h period does not increase the concentration of DNA synthesis stimulating activity that is observed in the medium. This activity is dialyzable and is resistant to acid treatment or to heating to 60-100 degrees C and to trypsin digestion; it is not extracted with chloroform/methanol nor adsorbed by charcoal or by a C18 reverse-phase column. Fractionation of the conditioned medium derived from Reuber H-35 hepatoma cells by gel filtration chromatography reveals two low molecular weight (less than 1000) compounds that both stimulate DNA synthesis in Fao hepatoma cells. The larger compound (peak I) also stimulates the activity of acetyl-CoA carboxylase. The stimulatory effects of the peak I compound are destroyed by nitrous acid deamination, periodate oxidation, and methanolysis. Biosynthetic labeling studies indicate the probable presence of glucosamine, galactose, and perhaps phosphate in the peak I-activating material. No significant incorporation of either myoinositol or mannose into the active material has been observed. These data, taken together, are consistent with a glycan structure for this autocrine factor, which bears strong resemblance to similar insulinomimetic factors generated in BC3H1 myocytes and H-35 hepatoma cells in response to insulin and on digestion of membranes with a phosphatidylinositol-specific phospholipase C. Further characterization of this factor may provide insight into different pathways of insulin action and could provide a strategy to check autocrine-stimulated tumor growth.

Acetyl-CoA Carboxylase↗

PubMed1988

"Tolerization" of human T-helper cell clones by chronic exposure to alloantigen: culture conditions dictate autocrine proliferative status but not acquisition of cytotoxic potential and suppressor-induction capacity.

Induction of clonal anergy in T-helper (Th) cells may have a role in regulating immune responses. A model system for studying Th cell tolerization at the clonal level in vitro could be useful for investigating the mechanisms involved. Accordingly, alloreactive helper cells were maintained in culture with interleukin 2 (IL 2) by intermittent stimulation with specific antigen. Regardless of the frequency of antigen stimulation, clones of age less than ca. 35 population doublings (PD) were found to undergo antigen-specific autocrine clonal expansion in the absence of exogenous IL 2. Such young clones (designated as phase I) could therefore not be "tolerized" by frequent exposure to antigen. In contrast, most clones of age greater than ca. 35 PD could be tolerized by frequent exposure to antigen (designated as phase II clones). Their autocrine proliferation was then blocked, although they still recognized antigen specifically as shown by their retained ability to secrete interferon-gamma (IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF). The mechanism of response failure involved both an inability to upregulate IL 2 receptors in the absence of exogenous IL 2, as well as an inability to secrete IL 2. These defects were not overcome by stimulation with mitogens or calcium ionophore and phorbol esther in place of alloantigen. T-cell receptor, alpha, beta, and gamma-chain gene rearrangements remained identical in phase I and phase II clones. Tolerization of phase II clones could be avoided by increasing the period between antigen exposures. Despite this, whether or not phase II cells were capable of autocrine proliferation, they were found to have acquired the novel function of inducing suppressive activity in fresh lymphocytes. Suppressor-induction was blocked by the broadly reactive MHC class II-specific monoclonal antibody (moAb) TU39, but not by moAb preferentially reacting only with HLA-DR, DQ, or DP. Sequential immunoprecipitation on T-cell clones showed the presence of a putative non-DR, DQ, DP, TU39+ molecule on phase II clones. However, this molecule was also found on phase I clones. The nature of the TU39-blockable suppressor-inducing determinant present on phase II but not on (most) phase I clones thus remains to be clarified. In addition to suppressor-induction activity, phase II clones also acquired lytic potential as measured in a lectin approximation system. Cytotoxic (CTX) potential was also not influenced by the frequency of antigenic stimulation and could be viewed as a constitutive modulation of clonal function.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1986

Autocrine stimulation by estradiol-regulated growth factors of rat hormone-responsive mammary cancer: interaction with the polyamine pathway.

We have recently shown that the integrity of the polyamine pathway is essential but not sufficient for expression of the mitogenic effect of estradiol (E2) in the N-nitrosomethylurea-induced rat mammary tumor grown in vitro in the soft agar clonogenic assay. To elucidate the mechanism of E2 action in this system, we tested whether E2 may stimulate tumor growth through induction of secretory growth factor(s), as already proposed for human breast cancer cell lines in culture. Furthermore, we investigated the potential interaction between such "autocrine" control of tumor growth by E2 and the polyamine pathway. Conditioned medium obtained from E2-treated tumors (E2-CM) but not from control tumors (C-CM) consistently stimulated colony formation when added to N-nitrosomethylurea-mammary tumors plated in soft agar under serum-free media conditions. Such growth-promoting effects of the E2-CM was found to increase with increasing protein concentrations of the medium and was abolished by pretreatment of the medium with concanavalin A, heat, and trypsin. The addition of the polyamine biosynthetic inhibitor alpha-difluoromethylornithine (1 mM) totally abolished the colony-stimulating effect of the E2-CM. Exogenous administration of spermidine (0.1 mM) reversed the inhibitory effect of alpha-difluoromethylornithine on colony formation and restored the action of the E2-CM. Although the addition of polyamines alone did not affect the number of colonies formed, the administration of spermidine was found to significantly enhance in a dose-dependent fashion the colony-stimulating effect of suboptimal concentrations of E2-CM. Attempts to identify the E2-inducible growth factor in the E2-CM and in N-nitrosomethylurea-mammary tumor specimens using monoclonal antibodies raised against the Mr 52,000 E2-inducible protein gave negative results. We conclude that autocrine stimulation of tumor growth by E2 is not limited to human breast cancer cell lines but also occurs in individual experimental mammary tumors grown in soft agar. Our results show that the polyamines must be present for the expression of this "autocrine" control of tumor growth by E2. Finally, the identity of the E2-induced growth factor operating in our system remains to be determined.

Animals↗

PubMed1988

Autocrine growth of a human T-cell line is inhibited by cyclosporin A.

The effect of cyclosporin A (CsA), a potent immunosuppressive agent, on a human T-cell line, IARC 301, which constitutively secretes interleukin-2 (IL-2) and expresses high-affinity IL-2 receptors, was investigated. We show that CsA inhibits IARC 301 cell growth. CsA also prevents the constitutive secretion of IL-2 in this T-cell line by blocking transcription of the IL-2 gene. If exogenous IL-2 is added together with CsA for 3 days, the cells grow as well as untreated controls. Thus, under such conditions, CsA inhibits IARC 301 growth by preventing its endogenous constitutive IL-2 synthesis. This demonstrates that IL-2 stimulates the proliferation of this cell line by an autocrine pathway, in agreement with our previous data. We also show for the first time, that CsA not only can inhibit IL-2 production of T cells upon activation, but that it can also prevent ongoing constitutive IL-2 synthesis of a T-cell line. Autocrine growth stimulation of tumor cells by cytokines has been demonstrated in a few cases. CsA inhibits synthesis of several cytokines. Probing for the autocrine growth of tumor cells by studying the effect of CsA and its reversibility by cytokines on their proliferation may be simple and useful.

Cell Division↗

PubMed1988

Autocrine production of pre-B-cell stimulating activity by a variety of transformed murine pre-B-cell lines.

Current evidence suggests that the proliferation and differentiation of normal pre-B-cells may be regulated by interactions with mesenchymally derived stromal cells. The nature of these cell-cell interactions has not yet been fully elucidated, but the involvement of pre-B-cell stimulating factors produced by various mesenchymal cell lines has recently been demonstrated in the murine system. In this model, transformed pre-B-cells differ from their normal counterparts in their acquisition of autonomous growth potential, as seen by an ability to be maintained in vitro in the absence of mesenchymal cell feeders. To test the hypothesis that autonomy might be associated with autocrine growth factor production, we tested the ability of a spontaneous pre-B-cell transformant (H9 cells) and two independently derived Abelson murine leukemia virus transformed pre-B-cell lines to produce pre-B-cell stimulating factors. All three lines released activities that stimulated the proliferation of themselves or H9 cells when cultured at low cell densities. One of the three transformed pre-B-cell lines could also substitute for mesenchymal feeders to stimulate normal pre-B-cells. Time course studies of an evolving Abelson murine leukemia virus transformant showed that the production of an autocrine pre-B-cell growth factor increased concomitantly with the acquisition of autonomous growth potential suggesting a relationship between these two phenotypic changes. Preliminary characterization of the growth factor responsiveness of H9 cells and the nature of the autostimulatory activity produced by this line suggested its nonidentity with any known hemopoietic growth factor. Activation of autocrine growth factor production appears to be a common event in the evolution of malignant pre-B-cells arising through different oncogenic mechanisms and may therefore be relevant to the pathogenesis of human acute lymphoid leukemia.

Abelson murine leukemia virus↗

PubMed1987

Quantitation of autocrine regulation of tumor growth: a general phenomenological model.

We present a model that considers autocrine regulation of tumor growth as a feedback control system. The model is general in that it can accommodate any growth curve which fits the data. We propose an algorithm for quantitation of the open loop gain parameter and we define the autocrine gain parameter; these parameters express the efficiency of the feedback mechanism. The model provides guidelines for design of experiments to measure the contribution of autocrine regulation to growth of biological systems, particularly tumors.

Animals↗