Autonomy in tumor cell proliferation

Medical Hypotheses (1990) 32.249-254 fQ Longman Group UK Ltd 1990

Autonomy M. CHIGIRA,

03069877/90/0032-0249if10.M

in Tumor

Cell Proliferation

K. NODA and H. WATANABE

Department of Orthopedic Surgery, Maebashi, Gunma, 371 Japan

Gunma University

School of Medicine, 3-39-22

Showa,

Abstract - Autonomous replication of tumor cells seems to be an essential factor in the definition of the malignant tumor itself, although tumor cell proliferation is, in general, controlled by the host response including immunological reactions and microenvironment. The ca se of the autonomy can hypothetically be classified into four categories as follow: (a) au tb”- and paracrine growth stimulation; (b) growth factor receptor abnormalities; (c) abnormal signal transduction; (d) self-incitement of ‘initiator-replicon’ system in DNA replication. These intracellular mechanisms may play important roles in the autonomy as shown in autocrine growth factors from the data obtained in protein-free cell culture. Hypothetically, negative regulation systems on the initiator-replicon may play roles of cell replication in multicellular organisms. Oncogene products and growth factors may affect this regulation system.

Introduction

Discussion

Self-replication, autonomous proliferation of a cell, is considered to be an essential goal in the autonomy of monads. However, in multicellular organisms, autonomous proliferation of each cell is strictly controlled. It is obvious that autonomy in growth is potentially very dangerous to the survival of the organism if not closely regulated as soon as the cells are no longer needed. False autonomy in self-replication is demonstrated only by sexual mechanisms. On the other hand, it appears that malignant tumors show autonomous growth in vivo. What is autonomous replication of cells in multicellular organisms? How do tumor cells show autonomy in growth? What controls the autonomous proliferation in the organisms? These questions have not been fully discussed yet.

Date Date

receive 17 October 1989 accepted 27 November 1989

Autocrine

stimulation

One of the most essential characters of malignant cells is uncontrolled growth (l-4), although increased cell replication could not be regarded as the sine qua non of malignancy (2). The major biologic characteristic of cancer is an alteration in the control of cellular metabolism including autonomous replication (l-3). In this regard, it is hypothesized that the growth of malignant cells is enhanced by a growth factor secreted by the cells themselves (5-8). This process has been termed ‘autocrine secretion’ (7, 8). Autocrine secretion of growth factors is a concept which is emerging as a unifying theme in the search for the molecular and cellular basis of malignant transformation (9-11). Tumor cells require fewer exogenous growth factors for optimal growth and multiplication than do their normal counterparts (8. 9). To explain this phenomenon, it was suggested that cells can become malignant by the

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endogenous production of polypeptide growth factors acting on their producer cells via functional external receptors, allowing phenotypic expression of the peptide by the same cell that produces it (8, 10). Actually, many of growth factors including insulin-like growth factors (IGFs) (11, 12), transforming growth factors (TGFs) (13-17), and several others (13,18-20) have been purified using serum-free (21-24) and proteinmedium (25-30). The necessity of free insulin-like growth factor for the in vivo growth of tumor cells has been shown recently (31). A phenomenological model of growth selfincitement has been proposed (32). On the other can confer growth factor hand, oncogenes autonomy on cancer cells (33-35). Deuel summarized the homology between growth factors and oncogene products (36). He stated that oncogene products appear to serve as autocrine growth factors. However, it is not clear what determines whether a cell is stimulated to grow normally for several cell cycles or alternatively, is transformed by oncogene products (36). it was reported that a virusFurthermore, transformed cell line (NPl) lacked detectable growth-promoting activity in conditioned media (36). The present authors established a murine fibrosarcoma cell line that needs no exogenous growth factors including TGFs, PDGF, IGF, insulin, transferrin, and hormones. Furthermore, insulin and transferrin were detected in the conditioned medium of the sarcoma (37). Complete protein-free medium is suitable for the culture of the line for long time (37). It has not been established whether intracellular oncogene products stimulate singnal transduction pathways (36). Receptor abnormalities

Holley concluded that the changes in a transformed cell that lead to lowered requirements for the growth factors may be the result of direct internal loss of control of DNA synthesis or membrane changes that modify the requirements for hormone interaction with the membrane (9). Enhanced cellular responsiveness to a growth factor may result also from a change in the number or affinity of the receptors for the growth factor (7, 8). Thus very large numbers of epidermal growth factor (EGF) receptors occur in squamous cell carcinoma (10, 38-42). A population of receptor molecules with extremely high affinity for EGF has been identified and may be involved in the mitogenic response of cells (43, 44). Downward showed a close similarity of 250

1IYPOTHESES

epidermal growth factor receptor and verb-B oncogene protein sequences (45). Another type of tumor cell in which a receptor abnormality seems to be important is the leukemic adult human T cell (7, 8). Unregulated expression of the receptor for interleukin-2 (IL-2) may contribute to leukemogenesis (46, 47), although at present there is little evidence for significant autocrine action of IL-2 itself in leukemic T cells (48). These observations have been made on the hypothesis that malignant transformation of cells is a kind of receptor disease. According to this hypothesis, receptor molecule abnormalities and their hyperproduction will be studied further. However, hormone receptor expression was not so prominent in carcinoma of the breast and prostate (49-51). Furthermore, hormone antagonists may not inhibit tumor growth in vivo (52, 53), since tumor cells unresponsive to hormone antagonists increase in number (53). In view of the data above, all of the autonomous tumor growth may not be due to the abnormalities of receptors for hormones and growth factors. Abnormal

signal transduction

A further way to achieve an enhanced degree of signaling by the pathway normally activated by either Transforming growth factor-alfa (TGFalfa) or EGF is to make the activation of the receptor independent of the effect; the erb-B oncogene may function in this manner to transform cells. The product of this gene has significant sequence homology to the transmembrane and cytoplasmic (tyrosine kinase) domains of the EGF receptor (35). Other oncogene products localized on the cell membrane (54), as well as other growth factor receptors, also have intrinsic tyrosine kinase activity (55-57). The p21 product of the Ha-ras gene is localized on the cytoplasmic side of the plasma membrane (58, 59) and binds GTP (60). The homology between the ras oncogene products and the G-protein regulators of adenyl cyclase activity (60-62) which require bound GTP for activity, implicate ras oncogene in transduction of the signal across the plasma membrane. Experiments showing the GTPbinding activity of transforming Ha-ras p21 to be enhanced by EGF (60-62) further suggest that secretion of TGF-alfa by ras-transformed cells has a role in amplifying this signaling pathway (7, 8). On the other hand, Huang demonstrated that reverse v-sis-mediated transformation with antisera to platelet-derived growth factor

.9117‘01\;OMY IN TlIMOK

CELL PROLIFERATION

(PDGF) has had limited success (63). Keating showed that activation of PDGF receptors in v-sis-transformed cells occurred in intracellular compartments, disrupting receptor processing and diverting receptors and their precursors to a chloroquine-sensitive degradation pathway (64). To date, the transduction system, including Gbinding protein, in these systems has only been discussed in a few reports (60-62). Elevated levels of cytosolic calcium and calcium binding proteins have been demonstrated by several authors (65-69). From these observations, postreceptor abnormalities may be summarized as follows; (a) abnormal transduction system so that the formation of the growth factor-receptor complex does not result in effective stimulation of the adenylate cyclase, (b) abnormal adenylate cyclase, (c) inactive phosphodiesterase, (d) protein kinase abnormalities, (e) abnormalities of enzymes or proteins that are substrates for this kinase. (f) phosphatases that antagonize protein reactions that kinase, and (g) biochemical mediate growth factor effects independent of cAMP. These summaries were based on the analysis of hormone responsiveness (70). Weinberg hypothesized that the mutated, oncogene versions of the transducing protein would seem to send out growth stimulatory signals even in the absence of prior stimulation from an upstream source such as autocrine growth,factors and receptor abnormalities (71). The da@ mentioned above do not clearly demonstrate that the primary signal of proliferation was transduced abnormally from the receptors to DNA. Recently, several direct evidences of signal transduction pathways have been reported. Kelvine showed that two signal transduction pathways distinguished by toxins are present in the stimulation by serum (72, 73). Increased activity of protein kinase C activity was reported in lung cancer (74). Matsumoto demonstrated that a protein kinase inhibitor interfered with cell proliferation (75). Initiator-replicon hypothesis It is questionable whether tumor autonomy is caused by the abnormalities of these signal transduction systems or not. The working hypothesis that the fundamental biochemical events which regulate cell division are similar in both bacteria and multicellular organisms has been accepted by many authors (76, 77). The most plausible model of DNA initiation is based on the repressor-operator model for regulation of protein synthesis through messenger RNA syn-

thesis (76). An entire DNA molecule is considered to be a unit of DNA replication. This is named a ‘replicon’ (76, 77). When the replicator gene receives this initiator protein, DNA synthesis is initiated and is propagated down the chromosome (77). The main conclusion was that initiation of DNA replication requires the synthesis of .a replicon-specific cytoplasmic protein (77). However, in the multicellular organisms, this ‘initiator-replicon’ hypothesis has not be tested. We have several questions on this hypothesis in cell replication. What chemical change occurs upon initiation? What is the biological role of growth factors, receptors. and signal transduction systems in the cell proliferation? What is the initiator? These questions have not been answered even in the microorganisms (77). One of the major differences between monads and multicellular organisms in self-replication may be that exogenous positive signals including growth factors and hormones is necessary in the multicellular organisms. Cell replication of the higher organisms are strictly regulated so that their cells are in a steady-state balance between duplication and destruction (l4, 77). On the other hand, self-replication of microorganisms is essentially autonomous. This difference between the monads and multicellular organisms should be analyzed further. It may be suggested that the ‘initiator-replicon’ system plays roles in the multicellular organisms from the data obtained by protein-free cell culture. It is important to find the initiator-replicon system, since no work on this field has been reported in the multicellular organisms. Hypothetical approach

We are within striking distance of organizing a disparate collection oncogenes, growth factors, receptors, and signal transduction mechanisms into a well-integrated coherent picture (71). Several authors have demonstrated that tumorigenesis is a multistep process and that a simple point mutational event is not easily reconciled with the observed complexity (78, 79). On a purely deductive basis. autocrine secretion should be viewed as an even more primitive use of ‘regulatory chemical messengers’ than either endocrine or paracrine secretion (7, 8). Autocrine mechanisms for self-stimulation would confer obvious selective growth advantages on very early embryonic cells and this could help to account for the explosive growth and multiplication of cells that occur in building a mass of cells that will survive as an organism (7. 8). However,

MEDICAL

hypothetically, it is obvious that the most primitive type of proliferation of cells is self-replication without any exogenous signals including autoand paracrine systems, as shown in monads. The most important question is why the exogenous positive stimulation of cell division is necessary in multicellular organisms. Is the exogenous negative regulation enough to control the ‘initiator-replicon’ system? There are two possibilities: the first possibility is a ‘loss of initiator-replicon’ in the multicellular organisms and the second is the intracellular negative regulation of initiator-replicon playing roles to control cell replication. Several reports have demonstrated that exogenous negative regulation plays roles to control cell proliferation (80-86) as shown in autocrine TGF-beta (87, 88) and EGF (89). However, it seems likely that the exogenous positive stimulation mentioned above is necessary in the cell proliferation of the multicellular organisms in general. It is unlikely that one of the most basic systems of organisms, initiatorreplicon, is lost in the multicellular organisms. We would like to propose that an intracellular negative regulation system on the ‘initiatcrreplicon’ plays an important role to control cell balance. Negative regulation of initiator production is essentially needed to balance the microenvironment in normal tissues of multicellular organisms. On this hypothesis, it is easy to explain the data that oncogenesis is multistep process (77-79), since oncogenesis may be considered as a step-wise loss of the negative regulation due to carcinogens and viral products. The autonomous tumor cell replication may be due to the complete loss of the intracellular negative regulation of the ‘initiator-replicon’ complex. This self-incitement may not be influenced by negative regulation (80-86). Under these premises, hormones and growth factors may enhance initiator production in cytoplasms by inhibiting the negative regulation. At least, the hypotheses including auto- and paracrine systems, receptor abnormalities, and abnormal transduction systems can not completely explain the tumor cell diversity described elsewhere (9094). The diversity should be considered as loss of control mechanisms of gene expression in general. Acknowledgement We thank Drs. Mitsuo Nagase, Tetsuya Shinozaki, Toru Shimizu, and Professor Eiichi Udagawa, Department of Orthopedic Surgery. Gunma University School of Medicine for their valuable discussion. This study was partially supported

HYPOTHESES

by a Scientific Research Grant (C) from the Ministry of Education, Science and Culture, Japanese Government.

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