HIV-1 Tat peptide immunoconjugates differentially sensitize breast cancer cells to selected antiproliferative agents that induce the cyclin-dependent kinase inhibitor p21WAF-1/CIP-1

In this study, we evaluated the ability of anti-p21 antibodies conjugated to 17-mer peptides [GRKKRRQRRRPPQGYGC] harboring the membrane-translocating and nuclear import sequences [underlined] of HIV-1 tat protein to inhibit the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1) (p21) and differentially sensitize MDA-MB-468 and MCF-7 human breast cancer (BC) cells to the antiproliferative effects of treatments that induce or do not induce p21. BC cells were treated with increasing concentrations of epidermal growth factor (EGF; 0.5-10 nM), the topoisomerase I inhibitor, camptothecin (CPT; 0.1-4 muM), or increasing doses of gamma-radiation (2-20 Gy). Western blot was used to evaluate p21 expression. The effect of treatment on cell cycle distribution was studied. Growth inhibition was measured by the WST-1 assay. Expression of p21 was increased in MDA-MB-468 cells treated with EGF or CPT but not by gamma-irradiation. MCF-7 cells exhibited p21 upregulation following exposure to CPT and gamma-radiation but not EGF. EGF caused cell cycle arrest in G(1) phase for MDA-MB-468 cells. CPT caused G(1)-phase arrest in MDA-MB-468 cells and prolonged S phase in MCF-7 cells. gamma-Radiation caused an increase in cells in G(2)/M phase for MDA-MB-468 and MCF-7. MDA-MB-468 cells were growth-inhibited by EGF, CPT, and gamma-radiation. MCF-7 cells were growth-stimulated by EGF and inhibited by CPT and gamma-radiation. Combining EGF with tat-anti-p21 immunoconjugates (ICs) amplified the growth-inhibitory effect on MDA-MB-468 cells 1.2-fold to 2.3-fold, but had no effect on the growth stimulation of MCF-7 cells by EGF. Tat-anti-p21 ICs sensitized MCF-7 cells 1.4-fold to gamma-radiation but had no effect on the growth of gamma-irradiated MDA-MB-468 cells. Tat-anti-p21 ICs sensitized both MDA-MB-468 and MCF-7 cells 1.7-fold to CPT. We conclude that tat-anti-p21 ICs are promising sensitizers for cytotoxic cancer therapies and that their sensitization is dependent on treatment-related p21 expression. This general approach could potentially be extended to other growth-regulatory molecules that are associated with tumor growth and progression.     

Induction of EGF-dependent apoptosis by vacuolar-type H(+)-ATPase inhibitors in A431 cells overexpressing the EGF receptor

The stimulation of human tumor cells overexpressing epidermal growth factor receptor (EGFR) with EGF enhances tumor development and malignancy. Therefore, compounds that modulate the EGF-mediated signal inducing apoptosis in EGFR-overexpressing cells would represent a new class of antitumor drug and might be useful in the treatment of a subset of human tumors. In the course of screening for compounds that induce apoptosis in EGFR-overexpressing human epidermal carcinoma A431 cells from secondary metabolites of microorganisms, we found that vacuolar-type H(+)-ATPase (V-ATPase) inhibitors, such as concanamycin B and destruxin E, induced apoptosis only when the cells were stimulated with EGF. The EGF-dependent apoptosis by V-ATPase inhibitors was not observed in other types of human tumor cells which do not overexpress EGFR. The apoptosis in A431 cells was inhibited by anti-FasL antibody which neutralized the cytotoxic effect of FasL, indicating that the Fas/FasL system was involved. The expression of cell surface FasL was upregulated by stimulation with EGF and increased further by V-ATPase inhibitors. Moreover, EGF inhibited cytotoxic Fas antibody-induced apoptosis, whereas V-ATPase inhibitors disrupted the protective effect of EGF on apoptosis in A431 cells. Taken together, these results suggested that V-ATPase inhibitors induced EGF-dependent apoptosis in A431 cells, possibly through both the enhancement of EGF-induced cell surface expression of FasL and the disruption of an EGF-induced survival signal.     

Structure based design and synthesis of peptide inhibitor of human LOX-12: in vitro and in vivo analysis of a novel therapeutic agent for breast cancer

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors, such as epidermal growth factor (EGF), with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid, which can be further metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) pathways to produce prostaglandins. The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression COX, LOX and other inflammatory enzymes. Ten peptides were designed and synthesized by solid phase peptide synthesis and analyzed in vitro for enzyme inhibition. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (K(D)) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.39 × 10(-8) M and 8.6 × 10(-8) M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.45 × 10(-7) M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC(50)) of 75 μM and 400 μM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 μM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p<0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.     

Signaling pathways regulating aromatase and cyclooxygenases in normal and malignant breast cells

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C(19) androgens to C(18) estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE(2) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression in breast cancer specimens. Knowledge of the signaling pathways that regulate the expression and enzyme activity of aromatase and cyclooxygenases (COXs) in stromal and epithelial breast cells will aid in understanding the interrelationships of these two enzyme systems and potentially identify novel targets for regulation. The effects of epidermal growth factor (EGF), transforming growth factor-beta (TGFbeta), and tetradecanoyl phorbol acetate (TPA) on aromatase and COXs were studied in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells MCF-10F, estrogen-responsive human breast cancer cells MCF-7, and estrogen-unresponsive human breast cancer cells MDA-MB-231. Levels of the constitutive COX isozyme, COX-1, were not altered by the various treatments in the cell systems studied. In breast adenocarcinoma cells, EGF and TGFbeta did not alter COX-2 levels at 24h, while TPA induced COX-2 levels by 75% in MDA-MB-231 cells. EGF and TPA in MCF-7 cells significantly increased aromatase activity while TGFbeta did not. In contrast to MCF-7 cells, TGFbeta and TPA significantly increased activity in MDA-MB-231 cells, while only a modest increase with EGF was observed. Untreated normal adipose stromal cells exhibited high basal levels of COX-1 but low to undetectable levels of COX-2. A dramatic induction of COX-2 was observed in the adipose stromal cells by EGF, TGFbeta, and TPA. Aromatase enzyme activity in normal adipose stromal cells was significantly increased by EGF, TGFbeta and TPA after 24h of treatment. In summary, the results of this investigation on the effects of several paracrine and/or autocrine signaling pathways in the regulation of expression of aromatase, COX-1, and COX-2 in breast cells has identified more complex relationships. Overall, elevated levels of these factors in the breast cancer tissue microenvironment can result in increased aromatase activity (and subsequent increased estrogen biosynthesis) via autocrine mechanisms in breast epithelial cells and via paracrine mechanisms in breast stromal cells. Furthermore, increased secretion of prostaglandins such as PGE(2) from constitutive COX-1 and inducible COX-2 isozymes present in epithelial and stromal cell compartments will result in both autocrine and paracrine actions to increase aromatase expression in the tissues.     

南方红豆杉和东北红豆杉中的单体化合物对乳腺癌细胞增殖的影响

本研究采用MTT法,研究南方红豆杉和东北红豆杉中的10种不同结构类型的单体化合物对乳腺癌MCF-7细胞增殖的影响。结果表明,化合物1~10(10-9~10-5mol/L)处理MCF-7细胞48和72 h后,仅化合物4在10-7、10-6和10-5mol/L浓度对MCF-7细胞增殖均有明显抑制作用,抑制率分别为29.8%、46.4%、51.8%和43.6%、61.2%、63.2%,与紫杉醇抑制细胞增殖的活性相近,且在24~72 h范围内具有时间依赖性;化合物2仅在10-5mol/L具有明显抑制细胞增殖的作用,抑制率为44.4%和49.6%。因此,10种不同结构类型的单体化合物中,仅baccatin III类化合物2、4对MCF-7细胞增殖具有较强的抑制作用,其中化合物4作用最强,活性与紫杉醇相近。     

Regulation of epidermal growth factor-receptor by estrogen and antiestrogen in the human breast cancer cell line MCF-7

Regulation of breast tumor proliferation depends in a large part on a variety of hormones and growth factors. In this report we show that estrogen and antiestrogen modulate epidermal growth factor-receptor (EGF-R) level in the human breast cancer MCF-7 cells with opposite mechanisms. Although a short-term treatment (24h to 48h) with estradiol leads to a decrease in EGF-R number, the addition of hormone in cell culture for 5 days increases EGF-R level with a maximal effect observed at 10(-10) M estradiol. In contrast, when cells are treated with the antiestrogen hydroxytamoxifen, a dose-dependent decrease in EGF-R level occurs. We also report that EGF is able to induce estrogen receptors and, to a lesser extent, progesterone receptors when added to MCF-7 cell cultures. These results demonstrate an interaction between both estrogen receptor and EGF receptor growth promoting systems in target cells. The implications of such an interaction in the understanding of human breast cancer hormone responsiveness and, in the development of therapies, are discussed.     

Electrochemical immunoassay on expression of integrin β1 on tumor cells and drug-resistant tumor cells

An electrochemical indirect competitive immunoassay protocol as a promising cytosensing strategy was developed to detect integrin β1 expression on human breast cancer MCF-7 cells and adriamycin-resistant human breast cancer MCF-7 (MCF-7/ADR) cells and quantify the cell number. Integrin α5β1 was adsorbed on the gold-nanoparticle modified glassy carbon electrode to bind integrin β1 monoclonal antibody (anti-CD29 mAb). A sandwich structure was then formed using nanocomposites which consisted of horseradish peroxidase (HRP) labeled anti-antibody and gold nanoparticles. HRP bound on the electrode surface could cause an amperometric response of the hydroquinone-H(2)O(2) system. The assembly of the sandwich structure was inhibited by tumor cells to give decreased enzyme-catalytic signals due to the capture of anti-CD29 mAb by integrin β1 on cell membranes. Under optimal conditions the relative current change (S) was proportional to the cell concentration from 1.6×10(3) to 2.0×10(6)cellsmL(-1) with a detection limit of 700cellsmL(-1). Integrin β1 expression in MCF-7/ADR cells was found to be significantly higher than that in MCF-7 cells, indicating the increased adhesion ability of MCF-7/ADR cells.     

EGF-dependent cell cycle progression is controlled by density-dependent regulation of Akt activation

The normal human breast epithelial cell line, MCF10A, was used to investigate the mechanism by which high-density inhibits EGF-dependent cell cycle progression. EGF-dependent Akt activation was found to be transient in high-density cells and sustained in low-density cells. High-density cells also showed decreased EGF receptor (EGFR) autophosphorylation, decreased retinoblastoma protein phosphorylation, and increased p27 protein expression. Although EGFR activation was decreased in the high-density cells, the activation was sufficient to stimulate EGFR substrates comparable to low-density cells. EGF-dependent activation of the Erk1/2 pathway and the upstream activators of Akt (Gab1, erbB3, PI3 kinase, and PDK1) showed no density dependency. Antagonists of Akt activity provided further evidence that regulation of Akt activation is the critical signal transduction step controlling EGF-dependent cell cycle progression. Both adenovirus-mediated expression of dominant-negative Akt and inhibition of PI3 kinase-mediated Akt activation with LY294002 blocked cell cycle progression of low-density cells. In summary, we report the novel finding that high-density blocks EGF-dependent cell cycle progression by inhibiting EGF signaling at the level of EGF-dependent Akt activation rather than at the level of EGFR activation.     

负载Her-2多肽的DCIK细胞对乳腺癌细胞杀伤作用研究

本文观察利用树突状细胞(DC)呈递肿瘤抗原(Her-2多肽)的特性提高DCIK细胞对乳腺癌细胞的杀伤活性。提取外周血来源的有核细胞诱导分离出细胞因子诱导的杀伤细胞(CIK)和树突状细胞(DC),DC负载Her-2多肽后和CIK细胞共培养产生DCIK细胞,并鉴定其HLA基因型。分析三株肿瘤细胞(MDA-MB-231、SK-BR-3、MCF-7)HLA基因型和Her-2蛋白表达情况。用细胞毒试验(CCK-8法)测定DCIK细胞的对三株Her2表达不同的乳腺癌细胞株的杀伤活性。结果表明DCIK细胞对MDA-MB-31、SK-BR-3、MCF-7的杀伤率(效靶比10:1)分别为50.38%±3.25%、52.19%±3.25%、47.09%±2.41%。而负载Her-2多肽的DCIK细胞对MDA-MB-231、SK-BR-3、MCF-7的杀伤率分别为76.30%±1.74%(P<0.001)、55.70%±3.05%(P=0.0143)、47.67%±2.40%(P=0.6972)。实验证明负载Her-2多肽的DCIK细胞能显著提高对Her-2( )的乳腺癌细胞的杀伤作用,为乳腺癌患者进行过继免疫治疗提供了理论依据。     

Human breast cancer cells synthesize and secrete an EGF-like immunoreactive factor in culture

A human breast cancer cell line, strain MCF-7, in culture synthesized and secreted a large amount of a polypeptide (designated as MCF-7 EGF) immunologically related to human epidermal growth factor (hEGF). The molecular weight of MCF-7 EGF estimated by gel filtration on Sephadex G-75 was similar to that of hEGF from human urine. On isoelectric focusing analysis, MCF-7 EGF gave a major peak at pH 4.6 and a minor peak at pH 5.0. In our enzyme immunoassay system, however, the dose-response curve of MCF-7 EGF did not show good parallelism with that of standard hEGF. From these results, it is suggested that MCF-7 cells synthesize and secrete a polypeptide immunologically related to hEGF into the culture medium.     

WISP-2/CCN5 is involved as a novel signaling intermediate in phorbol ester-protein kinase Calpha-mediated breast tumor cell proliferation

PMA and active phorbol esters stimulate the proliferation of various tumor cells, including ER-positive human breast tumor cell lines. However, the specific signaling pathways involved in the PMA-induced mitogenic effect on breast tumor cells have not been fully elucidated. In the present study, we explored the mechanisms associated with the mitogenic influence of PMA on breast tumor cells. Following an acute exposure (i.e., within 2 to 6 h) to PMA (50 nM), a mitogenic effect was observed on WISP-2/CCN5-positive breast tumor cell lines, including MCF-7, ZR-75-1 and SKBR-3 cells, and induction of WISP-2/CCN5 mRNA expression paralleled the observed mitogenic proliferation. This effect was undetected in WISP-2/CCN5 negative MDA-MB-231 breast tumor cells or human mammary epithelial cells with or without ER-alpha transfection. The mitogenic effect of PMA was perturbed by short hairpin RNA (shRNA)-mediated inhibition of WISP-2/CCN5 signaling in MCF-7 cells. Moreover, the upregulation of WISP-2/CCN5 by PMA is not ER dependent but is instead mediated through a complex PKCalpha-MAPK/ERK and SAPK/JNK signaling pathway, which leads to growth stimulation of MCF-7 breast tumor cells. These series of experiments provide the first evidence that WISP-2/CCN5 is a novel signaling molecule that critically participates in the mitogenic action of PMA on noninvasive, WISP-2/CCN5-positive breast tumor cells through PKCalpha-dependent, multiple molecular signal transduction pathways.     

Inhibition of mitogen-activated protein kinase kinase selectively inhibits cell proliferation in human breast cancer cells displaying enhanced insulin-like growth factor I-mediated mitogen-activated protein kinase activation.

Mitogen-activated protein (MAP) kinase mediates cell proliferation, cell differentiation, and cell survival by regulating signaling pathways activated by receptor protein tyrosine kinases (RPTKs), including the insulin-like growth factor 1 receptor (IGF-IR). We analyzed the upstream signaling components of the MAP kinase pathway, including RPTKs, in human breast cancer cell lines and found that some of those components were overexpressed. Importantly, signaling molecules such as IGF-IR, insulin receptor, and insulin receptor substrate 1, leading to the MAP kinase pathway, were found to be concomitantly overexpressed within certain tumor lines, i.e., MCF-7 and T-47D. When compared with the nonmalignant and other breast tumor lines examined, MCF-7 and T-47D cells displayed a more rapid, robust, and sustained MAP kinase activation in response to insulin-like growth factor I (IGF-I) stimulation. By contrast, IGF-I treatment led to a sustained down-regulation of MAP kinase in those lines overexpressing ErbB2-related RPTKs. Interestingly, blocking the MAP kinase pathway with PD098059 had the greatest antiproliferative effect on MCF-7 and T-47D among the normal and tumor lines tested. Furthermore, addition of an IGF-IR blocking antibody to growth medium attenuated the ability of PD098059 to suppress the growth of MCF-7 and T-47D cells. Thus, our study suggests that concomitant overexpression of multiple signaling components of the IGF-IR pathway leads to the amplification of IGF-I-mediated MAP kinase signaling and resultant sensitization to PD098059. The enhanced sensitivity to PD098059 implies an increased requirement for the MAP kinase pathway in those breast cancer cells, making this pathway a potential target in the treatment of selected breast malignancies.     

EGF-dependent epithelial cells of the mammary cell line HC11 demonstrate a high degree of synchronized cell cycle during stimulation with epidermal growth factor

The most popular object for studying endocytosis of EGF-receptor complexes, human epidermoid carcinoma A431, was shown to answer to EGF in high concentration (100 ng/ml) by growth inhibition, being indifferent to lower (0.1-1 ng/ml) concentrations. At the same time, cells NIH 3T3, expressing human EGF receptor (HER14), and epithelial mammary cells HC11 increased 14C-thymidine incorporation into DNA after EGF addition. However, for HER14 cells stimulatory effect of EGF was twice weaker than that induced by serum, whereas the effect of EGF on 14C-thymidine incorporation in DNA of cells HC11 was approximately 5 times stronger compared to serum. Therefore, cells HC11 may be referred to as EGF-dependent. Cell cycle analysis by fluorimetry showed that more than 90% of serum-starved HER14 and HC11 were in G0/G1. Within 19-20 h after stimulation by EGF 70-90% of HC11 cells and only 30-40% of HER14 cells were in S-phase. EGF removing from culture medium earlier than 9-11 h after stimulation blocked entering of HC11 cells into S-phase, whereas such EGF-dependent period was not found for cells HER14. Thus, synchronization of progression through early stages of cell cycle, stimulated by EGF and the presence of well defined "early" (EGF-dependent) and "late" (EGF-independent) phases, make cells HC11 convenient object for studying physiological role of EGF receptor complexes endocytosis.     

Angiopoietin-2, an angiogenic regulator, promotes initial growth and survival of breast cancer metastases to the lung through the integrin-linked kinase (ILK)-AKT-B cell lymphoma 2 (Bcl-2) pathway

The early onsets of breast cancer metastasis involve cell retention, survival, and resistant to apoptosis and subsequent growth at target vascular beds and tissues in distant organs. We previously reported that angiopoietin-2 (Ang2), an angiogenic regulator stimulates MCF-7 breast tumor metastasis from their orthotopic sites to distant organs through the α(5)β(1) integrin/integrin-linked kinase (ILK)/Akt pathway. Here, by using an experimental tumor metastasis model and in vitro studies, we further dissect the underlying mechanism by which Ang2 promotes the initial growth and survival of MCF-7 breast cancer metastasis in the lung of animals. We show that Ang2 increases cell survival and suppresses cell apoptosis through ILK-induced phosphorylation of Akt1, Akt2, and up-regulation of Bcl-2 in breast cancer cells. Inhibition of ILK, Akt1, and Akt2, and their effector Bcl-2 diminishes Ang2-stimulated breast cancer cell survival and Ang2-attenuated apoptosis in vitro, and initial survival and growth of breast cancer metastasis in the lung of animals. Additionally, siRNA knockdown of endogenous Ang2 in three human metastatic breast cancer cell lines also inhibits phosphorylation of Akt, expression of Bcl-2, and tumor cell survival, migration, and increases cell apoptosis. Since increased expression of Ang2 correlates with elevated potential of human breast cancer metastasis in clinic, our data underscore the importance that up-regulated Ang2 not only stimulates breast cancer growth and metastasis at late stages of the process, but is also critical at the initiating stages of metastases onset, thereby suggesting Ang2 as a promising therapeutic target for treating patients with metastatic breast cancer.     

A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15–30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.     

Growth of MCF-7 human breast carcinoma in severe combined immunodeficient mice: Growth suppression by recombinant interleukin-2 treatment and role of lymphokine-activated killer cells

Summary The severe combined immunodeficient (SCID) mouse, lacking functional T and B lymphocytes, has been considered by many groups to be a prime candidate for the reconstitution of a human immune system in a laboratory animal. In addition, this immuno-deficient animal would appear to have excellent potential as a host for transplanted human cancers, thus providing an exceptional opportunity for the study of interactions between the human immune system and human cancer in a laboratory animal. However, because this animal model is very recent, few studies have been reported documenting the capability of these mice to accept human cancers, and whether or not the residual immune cells in these mice (e.g. natural killer, NK, cells; macrophages) possess antitumor activities toward human cancers. Thus, the purpose of this study was (a) to determine whether or not a human breast carcinoma cell line (MCF-7) can be successfully transplanted to SCID mice, (b) to determine whether or not chronic treatment of SCID mice with a potent lymphokine (recombinant interleukin-2, rIL-2) could alter MCF-7 carcinoma growth, and (c) to assess whether or not rIL-2-activated NK cells (LAK cells) are important modulators of growth of MCF-7 cells in SCID mice. To fulfill these objectives, female SCID mice were implanted s.c. with MCF-7 cells (5 × 10⁶ cells/mouse) at 6 weeks of age. Six weeks later, some of the mice were injected i.p. twice weekly with rIL-2 (1 × 10⁴ U mouse^–1 injection^–1). Results clearly show that MCF-7 cells can grow progressively in SCID mice; 100% of the SCID mice implanted with MCF-7 cells developed palpable measurable tumors within 5–6 weeks after tumor cell inoculation. In addition, MCF-7 tumor growth was significantly (P <0.01) suppressed by rIL-2 treatment. rIL-2 treatment was non-toxic and no effect of treatment on body weight gains was observed. For non-tumor-bearing SCID mice, splenocytes treated in vitro with rIL-2 (lymphokine-activated killer, LAK, cells) or splenocytes derived from rIL-2-treated SCID mice (LAK cells) had significant (P <0.01) cytolytic activity toward MCF-7 carcinoma cells in vitro. In contrast, splenocytes (LAK cells) derived from tumor(MCF-7)-bearing rIL-2-treated SCID mice lacked cytolytic activities toward MCF-7 cells in vitro. No significant concentration of LAK cells in MCF-7 human breast carcinomas was observed nor did rIL-2 treatment significantly alter growth of MCF-7 cells in vitro. Thus, while rIL-2 treatment significantly suppressed growth of MCF-7 breast carcinomas in SCID mice, the mechanism of this growth suppression, albeit clearly not involving T and B lymphocytes, does not appear to be mediated via a direct cytolytic activity of LAK cells toward the carcinoma cells. However, rIL-2-activated SCID mouse splenocytes (LAK cells) do possess the capability of significant cytolytic activity toward MCF-7 human breast carcinoma cells. Thus, treatment of SCID mice with a potent lymphokine (rIL-2) induces a significant antitumor host response, a response that does not involve T and B lymphocytes and appears not to involve NK/LAK cells. This host response must be considered in future studies designed to investigate the interactions of reconstituted human immune systems and human cancers within this highly promising immuno deficient experimental animal model.     

Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.