Autocrine growth factors for human tumor clonogenic cells

A human epithelial-derived cell line, SW-13, releases a soluble substance that functions as an autocrine growth factor. SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, form a few small colonies when suspended in soft agar at low densities. The number of colonies increased significantly when either viable SW-13 cells or serum-free medium conditioned by SW-13 cells (CM) was added to agar underlayers. CM increased colony formation in a dose-dependent fashion. Clonal growth at low cell densities was dependent on the presence of both horse serum and SW-13 CM. Neither activity alone was capable of sustaining growth. Even when cells were plated at high densities CM could not substitute for serum, but could reduce the threshold serum concentration. The results suggest that autocrine and serum-derived factors act in concert to maintain clonal growth of epithelial tumor cells in soft agar.     

Autocrine and paracrine regulation by cytokines and growth factors in melanoma

Tumour development and progression involves the expression of oncogenes and inactivation of tumour suppressor genes, leading to the appearance of multiple malignant characteristics. Malignant melanoma cells express different growth factors and cytokines and their receptors in respective stages of tumour progression, which by autocrine and paracrine effects enable them to grow autonomously and confer competence to metastasis. Autocrine growth factors (bFGF, MGSA/GRO, IL-8 and sometimes IL-6, PDGF-A, IL-10) produced by melanoma cells stimulate proliferation of the producing cell itself, while paracrine growth factors (for example PDGF, EGF, TGF-beta, IL-1, GM-CSF, IGF-I, NGF, VEGF) modulate the microenvironment to the benefit of tumour growth and invasion. Paracrine effects include angiogenesis, stroma formation, modulation of host immune response, activation of proteolytic enzymes, adhesion or motility and metastasis formation. Some growth factors have inhibitory effects on melanocytes and early lesions (IL-1, IL-6, TGF-beta, OSM, TNF and IFN) but not on advanced stage melanomas, and in some cases they switch to autocrine stimulator (IL-6, TGF-beta). Understanding the involvement of different growth factors and cytokines in the molecular mechanism of melanoma progression will help to provide an insight into new future therapeutic approaches for melanoma.     

Polypeptide growth factors in embryogenesis and tumor growth

Polypeptide growth factors, which belong to different families (epidermal growth factors, insulin-like growth factors, fibroblast growth factors, transforming growth factors-beta, and some others), were characterized regarding their specific role in embryogenesis and tumor growth. Differences and parallels of the functioning of growth factors in these processes have been noted. Potential significance of the described characteristics of growth factors for directed modulation of embryogenesis and tumor growth is discussed.     

Blocking tumor cell eicosanoid synthesis by GP x 4 impedes tumor growth and malignancy

Using tumor cell-restricted overexpression of glutathione peroxidase 4 (GP x 4), we investigated the contribution of tumor cell eicosanoids to solid tumor growth and malignant progression in two tumor models differing in tumorigenic potential. By lowering cellular lipid hydroperoxide levels, GP x 4 inhibits cyclooxygenase (COX) and lipoxygenase (LOX) activities. GP x 4 overexpression drastically impeded solid tumor growth of weakly tumorigenic L929 fibrosarcoma cells, whereas B16BL6 melanoma solid tumor growth was unaffected. Yet, GP x 4 overexpression did markedly increase the sensitivity of B16BL6 tumors to angio-destructive TNF-alpha therapy and abolished the metastatic lung colonizing capacity of B16BL6 cells. Furthermore, the GP x 4-mediated suppression of tumor cell prostaglandin E(2) (PGE(2)) production impeded the induction of COX-2 expression by the tumor stress conditions hypoxia and inflammation. Thus, our results reflect a PGE(2)-driven positive feedback loop for COX-2 expression in tumor cells. This was further supported by the restoration of COX-2 induction capacity of GP x 4-overexpressing L929 tumor cells when cultured in the presence of exogenous PGE(2). Thus, although COX-2 expression and eicosanoid production may be enabled by PGE(2) from the tumor microenvironment, our results demonstrate the predominant tumor cell origin of protumoral eicosanoids, promoting solid tumor growth of weakly tumorigenic tumors and malignant progression of strongly tumorigenic tumors.     

Copper and zinc metabolism with solid tumor growth

The variation in copper and zinc metabolism with tumor growth appears to relate directly to progression or regression of the disease. Historically, elevations in serum copper have been used as clinical indicators in hematological neoplasms since the early 1960s. More recently, we have monitored breast, colo-rectal, and lung cancer patients for a six-month period through courses of cytotoxic chemotherapy to determine copper and zinc changes with tumor growth. Groups were divided into responders and nonresponders blind to their serum copper and zinc levels. Trends in elevated serum copper with active disease have shown similar trends in decreasing values with effective therapy, but normalization was at a slower rate. Serum zinc levels in the same patients were markedly below normal and did not increase in the study period. The clinical significance or elevated serum copper and depressed serum zinc is discussed and the potential relationship between the two elements is explored. A solid tumor-bearing rat model, mammary adenocarcinoma R 3230 AC, has detailed more of the changes in copper and zinc metabolism with solid tumor growth. Serum copper and zinc varied with tumor mass, as in clinical studies. Liver values of the two essential metals did not change significantly, but liver-related copper-containing enzymes showed marked variations. Ceruloplasmin in serum increased with increasing tumor mass, as would be expected with the increased serum copper levels. Cytochrome c oxidase activity in liver homogenates from tumor-bearing animals was significantly depressed.     

Autocrine growth limitation of human papillomavirus type 16-harboring keratinocytes by constitutively released tumor necrosis factor-alpha.

TNF-alpha is known to exert antitumor and antiviral effects and to participate in the regulation of the immune response. In our study we demonstrate that human rTNF-alpha specifically blocks growth of SK-v keratinocyte cell line harboring and expressing human papillomavirus type 16 (HPV16) sequences. This inhibitory effect was shown by [3H]TdR incorporation and cell counting. Binding experiments with 125I-TNF-alpha showed that SK-v cells express about 10,000 single class TNF-alpha R per cell with affinity constant of about 0.7 nM. Binding of 125I-TNF-alpha could be inhibited by htr-9 mAb recognizing a 55/60-kDa type I TNF-alpha R but not by utr-1 mAb recognizing 75/80-kDa type II TNF-alpha R or irrelevant mAb specific for HPV16E7 protein. Addition of anti-TNF-alpha antibodies to SK-v cell culture resulted in significant (p < 0.05), dose-dependent stimulation of their proliferation. SK-v cells constitutively expressed TNF-alpha mRNA, and SK-v CM contained TNF-alpha, as demonstrated by Northern blot analysis, a specific ELISA, Western blot analysis, and a bioassay with TNF-alpha-sensitive L-M cells. HPLC gel filtration of SK-v cell CM showed that the factor cytotoxic for L-M cells coeluted with immunoreactive TNF-alpha. These results demonstrate that HPV16-harboring SK-v cells constitutively express and release immunoreactive and biologically active TNF-alpha that in turn may exert an autocrine growth inhibitory effect. This phenomenon could represent one of the self-limiting mechanisms controling growth of HPV-induced neoplasia.     

Autocrine growth and progression of murine X-ray-induced T cell lymphomas.

Primary tumors of X-ray-induced murine T cell lymphomas comprise autocrine, growth factor-dependent cells. We have grown cell lines from primary X-ray-induced thymic lymphomas (PXTLs) under conditions which minimize the progression of the cells from factor dependence to factor independence. All (22) PXTL lines grown secrete a growth factor which supports their own growth and which we will call lymphoma growth factor LGF. LGF-dependent cells are non-tumorigenic or poorly tumorigenic, do not clone in soft agar, have no detectable rearrangements in the c-myc or Pim-1 region and possess near diploid or pseudodiploid karyotypes without evidence for trisomy of chromosomes nos. 15 or 17. PXTL-secreted LGF has no interleukin 1, 2, or 3 activity nor do LGF-secreting cells synthesize detectable IL-1, -2, or -3 mRNA. LGF contains no detectable interferon or GM-CSF activity in specific bioassays. Purified EGF, TGF beta, and interleukin preparations are inactive on LGF-dependent PXTL cells. Thus LGF appears to be a new growth factor that is required for the proliferation of non-progressed T lymphoma cells. Upon progression PXTL cells become growth factor independent, are highly tumorigenic in vivo, clone in soft agar, and assume a near triploid karyotype containing numerous chromosomal aberrations. Thus in X-ray-induced lymphomagenesis an autocrine, LGF-dependent phase precedes the progressed phase characterized by rearrangements in the myc and/or Pim-1 regions as well as by many chromosomal aberrations visible in the karyotype.     

Polyomavirus middle tumor antigen increases responsiveness to growth factors.

The middle tumor antigen (mT) of polyomavirus is unable to transform a clone of NIH 3T3 cells to anchorage independence (L. Raptis and J.B. Bolen, J. Virol. 63:753-758, 1989). However, this oncogene increased the responsiveness of these cells to the growth factors (alpha-like and beta-type transforming growth factors) produced by cells possessing the whole transforming region of polyomavirus. This resulted in the growth of NIH 3T3 cells, expressing mT under control of the dexamethasone-regulatable mouse mammary tumor virus promoter, in agar medium supplemented with these growth factors upon addition of the inducer. Therefore, mT, a transforming oncogene, is able to enhance the responsiveness of established cells to growth factors, a property previously attributed primarily to myc and other establishment type oncogenes.     

Platelet-derived growth factor in human malignancy.

Platelet-derived growth factor (PDGF) was first implicated in the process of transformation when one of its peptide chains was found to be homologous to the viral sis oncogene (v-sis). Since that time, there have been multiple demonstrations of the transforming activity of v-sis in fibroblasts. Because of the near identity of the v-sis protein with the PDGF B chain, v-sis is thought to transform through an autocrine stimulatory mechanism of cell growth. Consistent with this view are studies which demonstrate inhibition of v-sis-mediated transformation by anti-PDGF antibodies. Expression of the cellular sis gene (c-sis) and its receptors, and secretion of PDGF-like factors have been demonstrated in many types of human malignant cells. Nevertheless, a causative role for c-sis in inducing or maintaining the transformed phenotype in human malignancies remains to be established. There are significant differences in structure between v-sis and c-sis. Studies of transforming ability have yielded conflicting results in transfection models, depending on the transfected vector and target cell type utilized. While there is compelling evidence for the involvement of PDGF in an autocrine growth mechanism in transformed fibroblasts, the evidence in human epithelial tumor types is less convincing because PDGF receptors are usually not detectable on the cell surface. The recent demonstration of intracellular co-localization of active PDGF precursors and PDGF receptors, however, supports the existence of an internal autocrine pathway independent of PDGF secretion. Further investigation of such a mechanism in de novo human malignancies is warranted to establish the role of PDGF in the development of these neoplasms.     

Autocrine functions of VEGF in breast tumor cells

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.     

Autocrine stimulation of mas oncogene leads to altered growth control.

Mas oncogene has been shown to have focus-inducing ability in NIH 3T3 cells which are tumorigenic in vivo in nude mice. Its stable expression in a variety of cell lines conferred some angiotensin responsiveness. To understand why mas-transfected cells exhibit a transformed phenotype and if angiotensin responsiveness plays any role in this process, we studied the growth characteristics of mas-transfected 3T3 cells and demonstrated that they lose contact inhibition, exhibit foci formation, and increased DNA synthesis even in absence of serum. Our results suggest that the transformed phenotype is due to the production of a mas receptor ligand distinct from angiotensin.     

Autocrine and paracrine growth regulation of human breast cancer

Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vitro. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells, facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation, hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation.     

Autocrine growth hormone: effects on growth hormone receptor trafficking and signaling

GH and GH receptor are expressed in many extrapituitary tissues, permitting autocrine/paracrine activity. Autocrine GH has regulatory functions in embryonic development and cellular differentiation and proliferation and is reported to be involved in the development and metastasis of tumor cells. To understand the principles of transport and signaling of autocrine GH and GH receptor, we used a model system to express both proteins in the same cell. Our experiments show that GH binds the GH receptor immediately after synthesis in the endoplasmic reticulum and facilitates maturation of GH receptor. The hormone-receptor complexes arrive at the cell surface where exogenously added GH is unable to bind these receptors. Autocrine GH activates the GH receptors, but signal transduction occurs only after exiting the endoplasmic reticulum. This model study explains why autocrine GH-producing cells may be insensitive for GH (antagonist) treatment and clarifies autocrine signaling events.     

Autocrine growth factors secreted by the malignant human B-cell-line BJAB are distinct from other known cytokines

BJAB, a EBV-negative Burkitt-like lymphoma, did not grow under suboptimal culture conditions in low concentrations of serum unless appropriate cytokines were added. A subclone of BJAB, Clone 13, however, could be kept in long-term culture under such conditions without added cytokines. This suggested that growth of BJAB-Clone 13 was supported by autocrine growth factors (AGF). In fact, the supernatant of Clone 13 stimulated growth of the parental BJAB line and showed IL-1-like activity. Of several cytokines tested only AGF and IL-1 stimulated growth of BJAB. IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, GM-CSF, TNF-alpha, LT, IFN-gamma and TGF beta did not have this effect. The IL-1-like activity was completely neutralized by anti-IL-1 alpha antibodies. In contrast, AGF-activity was not affected by anti-IL-1 alpha. Rabbit antibodies produced against fractions enriched for AGF inhibited growth of BJAB. This inhibition was overcome by Clone 13-AGF, but not by IL-1 alpha. These data suggest that Clone 13-AGF is distinct from IL-1 alpha and might be a new cytokine.     

Prediction of ovarian tumor malignancy

Ovarian cancer is the leading cause of mortality among gynecological cancers. The aim of the study was to form the decision rules for distinguishing benign from malignant ovary lesions. The research was conducted on 201 women with ovary tumor. Commonly used specific markers for ovarian cancer (biochemical marker Ca 125, ultrasound and vascular markers) were used. The significant difference in the presence of an ultrasound and vascular markers between benign and malignant ovary changes along with the significantly different level of Ca 125 is confirmed. To a specific marker certain score number was appointed and the scoring system was formed. The incidence of benign/malignant ovary changes was observed in the researched group regarding anthropometric parameters (age, marital and menopausal status and number of deliveries). There is also significant difference in the incidence of benign/malignant ovary tumor regarding these parameters. Based on combination of the scoring system and anthropometric parameters the decision rules for distinguishing benign from malignant ovary tumors were formed. The logistic regression method was used. We proved that this method has higher accuracy in prediction of malignancy in women with ovary tumors than using morphological, Doppler or anthropometric parameters separately.     

Fetal antigens as tumor growth enhancing factors in Yoshida's tumor rats

Malignant neoplastic cells have been shown to have some antigenic features identical to those of embryonic cells. Since several antigens are likely to be shared by both embryonic cells and neoplastic tissue, we tried to understand the meaning of the appearance of such antigens and the type of effect that the immunization with embryonic antigens would have on the survival of Yoshida's tumor rats. Wistar rats were immunized with fetal antigens by fetal cells (1.5 x 10(6)) suspended in 0.5 ml of Hanks solution plus an equal volume of Freund adjuvant, were injected in hind footpads, i.p. and i.m., respectively, for active immunization. Rabbit antigen sera were used for passive immunization. All animals presented ascites and tumor growth. Animals immunized by means of fetal cell antigens showed a mean survival rate after neoplastic transplant of 14 days. Animals that received rabbit immune serum showed a mean survival rate after neoplastic transplant of 17 days. The immunization by means of fetal antigens elicited a scanty effect on the survival of Yoshida's tumor transplanted rats. It can be concluded that antibodies, which are able to cross react with neoplastic cells, do not have cytotoxic effect and do not interfere with the survival of the neoplastic transplanted animals. Therefore, fetal antigens are likely able to carry out an immunosuppressive action. The fact that they appear on neoplastic cells could be seen as a metabolic modification effect or as a growth enhancing factor.