Update of alpha fetoprotein growth-inhibitory peptides as biotherapeutic agents for tumor growth and metastasis.

The present update describes the biological activities of an alpha fetoprotein (AFP)-derived peptide termed the growth-inhibitory peptide (GIP), which is a synthetic 34-amino acid segment produced from the native molecule. The GIP has been shown to be growth-suppressive in both fetal and tumor cells but not in adult cells. Even though its mechanism of action has not been completely elucidated, GIP has been shown to engage in cellular events such as endocytosis, cellular aggregation, hemagglution, and cytoskeleton-induced cell shape changes. The GIP was shown to be growth-suppressive in nine different human tumor types and to suppress the spread of tumor infiltrates and metastases in human and mouse mammary cancers. It was further found that the oligomeric state (cyclic vs. linear configuration) of the GIP determined its biological and anticancer efficacy. The combined properties of tumor growth suppression and reduction of tumor metastases represent promising areas for GIP as a chemotherapeutic agent. It was concluded that the GIP derived from full-length AFP represents a growth-inhibitory motif possessing intrinsic properties that allow it to interact in cell surface events such as tumor proliferation, progression, and metastasis.

Review of Growth Inhibitory Peptide as a biotherapeutic agent for tumor growth, adhesion, and metastasis.

This review surveys the biological activities of an alpha-fetoprotein (AFP) derived peptide termed the Growth Inhibitory Peptide (GIP), which is a synthetic 34 amino acid segment produced from the full length 590 amino acid AFP molecule. The GIP has been shown to be growth-suppressive in both fetal and tumor cells but not in adult terminally-differentiated cells. The mechanism of action of this peptide has not been fully elucidated; however, GIP is highly interactive at the plasma membrane surface in cellular events such as endocytosis, cell contact inhibition and cytoskeleton-induced cell shape changes. The GIP was shown to be growth-suppressive in nine human tumor types and to suppress the spread of tumor infiltrates and metastases in human and mouse mammary cancers. The AFP-derived peptide and its subfragments were also shown to inhibit tumor cell adhesion to extracellular matrix (ECM) proteins and to block platelet aggregation; thus it was expected that the GIP would inhibit cell spreading/migration and metastatic infiltration into host tissues such as lung and pancreas. It was further found that the cyclic versus linear configuration of GIP determined its biological and anti-cancer efficacy. Genbank amino acid sequence identities with a variety of integrin alpha/beta chain proteins supported the GIP's linkage to inhibition of tumor cell adhesion and platelet aggregation. The combined properties of tumor growth suppression, prevention of tumor cell-to-ECM adhesion, and inhibition of platelet aggregation indicate that tumor-to-platelet interactions present promising targets for GIP as an anti-metastatic agent. Finally, based on cholinergic studies, it was proposed that GIP could influence the enzymatic activity of membrane acetylcholinesterases during tumor growth and metastasis. It was concluded that the GIP derived from full-length AFP represents a growth inhibitory motif possessing instrinsic properties that allow it to interfere in cell surface events such as adhesion, migration, metastasis, and aggregation of tumor cells.

Human alpha-fetoprotein contains potential heterodimerization motifs capable of interaction with nuclear receptors and transcription/growth factors.

Alpha-fetoprotein (AFP) serum levels in man have long been utilized as a tumor marker and as a birth defect screening agent in the clinical laboratory. Although the physiological role of AFP has remained obscure, the stereotypic carrier/transport function of a fetal counterpart to albumin has been attributed to this oncofetal protein. However, reports from a multitude of investigators in the last decade have provided a rationale to reconsider and extend the biological role of AFP to include cell growth modulation during development. Previously, a leucine zipper-like (heptad) motif, which mimicked that found in the steroid/thyroid receptor superfamily, was postulated for portions of the third domain of AFP. The present report proposes the presence of additional potential heterodimerization sites for the nuclear receptor superfamily members and other growth factors in the second and third domains of human AFP.

Five-chlorodeoxycytidine, a tumor-selective enzyme-driven radiosensitizer, effectively controls five advanced human tumors in nude mice.

PURPOSE: The study's goals were as follows: (1) to extend our past findings with rodent tumors to human tumors in nude mice, (2) to determine if the drug protocol could be simplified so that only CldC and one modulator, tetrahydrouridine (H4U), would be sufficient to obtain efficacy, (3) to determine the levels of deoxycytidine kinase and dCMP deaminase in human tumors, compared to adjacent normal tissue, and (4) to determine the effect of CldC on normal tissue radiation damage to the cervical spinal cord of nude mice. METHODS AND MATERIALS: The five human tumors used were as follows: prostate tumors, PC-3 and H-1579; glioblastoma, SF-295; breast tumor, GI-101; and lung tumor, H-165. The duration of treatment was 3-5 weeks, with drugs administered on Days 1-4 and radiation on Days 3-5 of each week. The biomodulators of CldC were N-(Phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartyl transcarbamoylase, 5-fluorodeoxycytidine (FdC), resulting in tumor-directed inhibition of thymidylate synthetase, and H4U, an inhibitor of cytidine deaminase. The total dose of focused irradiation of the tumors was usually 45 Gy in 12 fractions. RESULTS: Marked radiosensitization was obtained with CldC and the three modulators. The average days in tumor regrowth delay for X-ray compared to drugs plus X-ray, respectively, were: PC-3 prostate, 42-97; H-1579 prostate, 29-115; glioblastoma, 5-51; breast, 50-80; lung, 32-123. Comparative studies with PC-3 and H-1579 using CldC coadministered with H4U, showed that both PALA and FdC are dispensable, and the protocol can be simplified with equal and possibly heightened efficacy. For example, PC-3 with X-ray and (1) no drugs, (2) CldC plus the three modulators, (3) a high dose of CldC, and (4) escalating doses of CldC resulted in 0/10, 3/9, 5/10, and 6/9 cures, respectively. The tumor regrowth delay data followed a similar pattern. After treating mice only 11/2 weeks with CldC + H4U, 92% of the PC-3 tumor cells were found to possess CldU in their DNA. The great majority of head-and-neck tumors from patient material had markedly higher levels of dC kinase and dCMP deaminase than found in adjacent normal tissue. Physiologic and histologic studies showed that CldC + H4U combined with X-ray, focused on the cervical spinal cord, did not result in damage to that tissue. CONCLUSIONS: 5-CldC coadministered with only H4U is an effective radiosensitizer of human tumors. Ninety-two percent of PC-3 tumor cells have been shown to take up ClUra derived from CldC in their DNA after only 11/2 weeks and 2 weeks of bolus i.p. injections. Enzymatic alterations that make tumors successful have been exploited for a therapeutic advantage. The great electronegativity, coupled with the relatively small Van der Waal radius of the Cl atom, may result in CldC's possessing the dual advantageous properties of FdC on one hand and BrdU and IdU on the other hand. These advantages include autoenhancing the incorporation of CldUTP into DNA by not only overrunning but also inhibiting the formation of competing TTP pools in tumors. A clinical trial is about to begin, with head-and-neck tumors as a first target of CldC radiosensitization.

IRIDIUM exposure increases c-fos expression in the mouse brain only at levels which likely result in tissue heating.

With the rapid development of wireless communication technology over the last 20 years, there has been some public concern over possible health effects of long-term, low-level radiofrequency exposure from cellular telephones. As an initial step in compiling a database for risk analysis by government agencies, the effects of 1-h exposure of mice to a 1.6-GHz radiofrequency signal, given as either a continuous wave or pulse modulated at 11 Hz with a duty cycle of 4:1 and a pulse duration of 9.2 ms IRIDIUM), on c-fos gene expression in the brain was investigated. The IRIDIUM signal is the operating frequency for a ground-to-satellite-to-ground cellular communications web which has recently become fully operational, and was named as such due to the original designed employment of the same number of low orbiting satellites as there are electrons orbiting the nucleus of an iridium atom. The expression of c-fos was not significantly elevated in the brains of mice until exposure levels exceeded six times the peak dose and 30 times the whole body average dose as maximal cellular telephone exposure limits in humans. Higher level exposure using either continuous wave (analog) or IRIDIUM signals elevated c-fos to a similar extent, suggesting no obvious pulsed modulation-specific effects. The pattern of c-fos elevation in limbic cortex and subcortex areas at higher exposure levels is most consistent with a stress response due to thermal perception coupled with restraint and/or neuron activity near thermoregulatory regions, and not consistent with any direct interaction of IRIDIUM energy with brain tissue.

Delayed type hypersensitivity in patients with rheumatoid arthritis.

Our study examines the relationship between in vivo delayed type hypersensitivity (DTH) and clinical and laboratory variables associated with rheumatoid arthritis (RA). Eleven patients with RA were examined. They were receiving no disease modifying drugs, immunosuppressive agents or corticosteroids. Skin tests were performed with the Multitest CMI system employing 7 antigens simultaneously. No patients were truly anergic, however, 3 of the 11 patients were hyporesponsive. No difference was noted between the total in vivo DTH responses of the patient and control populations. A significant correlation was observed between total DTH and the overall activity of the RA as measured by the erythrocyte sedimentation rate (ESR). However, there was no relationship between the total DTH scores and the degree of synovitis. There was no correlation between DTH and a variety of laboratory variables including the autologous mixed lymphocyte response, and the percentage of activated CD4+ or CD8+ T lymphocytes. These observations indicate that while anergy was uncommon in our population of patients with RA, decreased DTH responses were associated with greater disease activity as determined by the ESR.

Signal transduction in Sjögren's syndrome T cells. Abnormalities associated with a newly described human A-type retrovirus.

OBJECTIVE: To study the effects of a novel A-type retrovirus, detected in cocultures of lip biopsy specimens from Sjögren's syndrome (SS) patients and a human T cell line, on the infected T cells. METHODS: Interleukin-2 (IL-2) and IL-6 secretion were measured by bioassay and enzyme-linked immunosorbent assay, respectively, in the infected and noninfected cell lines. Surface antigen expression was determined by flow cytometry, using monoclonal antibodies. Protein kinase C (PKC) activity was measured using an enzyme assay kit, and calcium mobilization was assessed with a fluorescent probe. RESULTS: Infected cells expressed less CD4 and IL-6 receptor, but more HLA-DR, compared with noninfected cells. Infected cells also produced less IL-2 and displayed reduced PKC activation and calcium mobilization. A similar defect in calcium mobilization was detected in T cells from SS patients. CONCLUSION: These data suggest a possible involvement of the newly described retrovirus in T cell abnormalities.

Detection of serum antibodies to retroviral proteins in patients with primary Sjögren's syndrome (autoimmune exocrinopathy).

Primary Sjögren's syndrome (SS) is considered a benign autoimmune disease; it is characterized by lymphoid infiltration of salivary and lacrimal glands, often accompanied by the presence of serum autoantibodies, particularly anti-Ro (SS-A) and anti-La (SS-B). There are important immunologic similarities between primary SS and acquired immunodeficiency syndrome. To investigate for a possible immune response to retroviral proteins in primary SS, we performed immunoblotting against human immunodeficiency virus-1 (HIV-1) proteins using sera from 47 patients with primary SS. Moderate-to-strong reactivity, suggesting the presence of serum antibodies, was found in 14 patients (30%). Of 120 normal subjects, only 1 showed moderate positivity. All 14 positive SS sera reacted against p24 (gag) but failed to react against gp41 or gp120 (env). This response did not reflect hypergammaglobulinemia since immunoglobulin concentrations among the 29 SS patients studied were the same in sera that contained and sera that did not contain anti-gag reactivity. Two sera also reacted against p17 gag. Four reacted against HIV-2 core proteins, but none reacted with core proteins of human T lymphotropic virus-I. Only 1 of the 14 sera reacted against Ro (SS-A), and 1 other reacted against La (SS-B). These results identify a subset of SS patients characterized by 1) the presence of serum antibodies to HIV-1 group-specific, but not type-specific, proteins, and 2) the relative absence of anti-Ro (SS-A) and anti-La (SS-B) autoantibodies. In this latter respect, these SS patients constitute a subpopulation that resembles patients with HIV-induced SS-like disease.

A conserved idiotype and antibodies to retroviral proteins in systemic lupus erythematosus.

22 of 61 systemic lupus erythematosus (SLE) patients produced antibodies to the p24 gag protein of HIV-1 demonstrated by Western blotting. 20 of these 22 patients (91%) also express the 4B4 idiotype (Id 4B4) previously identified on a human anti-Sm monoclonal antibody called 4B4. This represents an enrichment for this Id (seen in only 52% of SLE patients generally). Eight of these 22 SLE patients also have anti-Sm antibody activity. Sm partially inhibits the antibody binding of p24 gag suggesting immunologic cross-reactivity between the retroviral antigen p24 gag and the autoantigen Sm. Anti-Id 4B4 also inhibits p24 gag antibody binding by as much as 40%. Finally the monoclonal antibody 4B4 showed cross-reactivity to Sm and p24 gag. The following points emerge from our studies: (a) SLE patients make antibodies to p24 gag of HIV-1, (b) there is a relationship between immunity to p24 gag and a conserved idiotype, and (c) anti-Sm antibodies can cross-react with p24 gag.

Anti-peptide antibodies detect a lupus-related interspecies idiotype that maps to H chain CDR2.

Antibodies to the small nucleoprotein Sm occur spontaneously in human and murine systemic lupus erythematosus. Human and mouse monoclonal anti-Sm autoantibodies designated 4B4 and Y2 share an idiotype (Id) determinant located on the Ig H chain. To understand the molecular basis of this cross-reactivity, the VH regions of both antibodies were sequenced and analyzed for homology. The antibodies showed only 49.6% homology. The second complementary determining region (CDR2) was the most likely candidate for the Id site. To investigate this possibility, rabbit antiserum was made against a peptide corresponding to the CDR2 of 4B4. This antiserum was specific for the immunizing peptide and reacted weakly to a peptide corresponding to the CDR2 of Y2. Anti-CDR2 antibody bound to 4B4 and Y2 but not to other human and mouse mAb. Binding was directed at the H chain when analyzed by Western blots. Anti-CDR2 antibody blocked anti-Id antibody binding to 4B4 and Y2 by 58% and 24%, respectively. These studies suggest that this interspecies Id maps to the H chain CDR2 and that a conserved Id can occur within molecules that are otherwise radically different.

Characterization of the IL-2-receptor on rheumatoid arthritis synovial fluid T cells.

We studied the hypoproliferative response of synovial fluid (SF) T cells in rheumatoid arthritis (RA) using a mitogenic monoclonal antibody (MoAb) specific for the T-cell antigen receptor-associated CD3 complex. RASF T cells are defective in their proliferative response and in the induction of the Tac (p55) component of the IL-2-receptor (IL-2-R) when stimulated with anti-CD3 monoclonal antibody (MoAb). However, fresh RASF T cells bear demonstrable IL-2-R in cross-linking experiments which are not seen in unstimulated peripheral blood (PB). These receptors are functional since RASF T cells proliferate in response to recombinant IL-2 (rIL-2) better than fresh PB T cells from either normal or RA patients. Scatchard analysis indicates increased (4-fold) numbers of high affinity IL-2-R on (phytohaemagglutinin) PHA-activated RASF T cells as compared with comparably activated RAPB T cells. Phorbol myristate acetate (PMA) induces Tac antigen expression in RASF but does not lead to proliferation. The hyporesponsiveness of RASF T cells does not appear to result from lack of IL-2-R, lack of IL-2-R inducibility, or proliferative potential.

Autostimulatory growth factors produced by Sjögren's syndrome B cell lines.

Sjögren's syndrome (SS) is a systemic autoimmune disorder characterized by polyclonal hypergammaglobulinemia, autoantibody formation, and intense tissue infiltration by lymphoid and plasma cells. These patients have a predisposition to the development of monoclonal immunoglobulins and B cell lymphomas. To study the role of B cells in this disease, we established B cell lines from three patients with benign primary SS. These B cell lines grew spontaneously without any stimulation and expressed EBV nuclear antigen. The lines secreted an autostimulatory factor which had properties of a B cell growth factor. Peripheral blood B cells from SS patients, after culture for 3 days, secreted a similar factor which stimulated the proliferation of established SS B cell lines. These results suggest that circulating B cells in SS produce an autocrine growth factor that may contribute to lymphoproliferation and ultimately to the emergence of B cell lymphomas.

Autostimulatory factor produced by B-cell lines from patients with Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease with a predisposition to transform into a B-cell lymphoma. Stable B-cell lines were established (without exogenous stimulation other than fetal bovine serum) from the peripheral blood of three SS patients. These cell lines secreted immunoglobulin (either IgG, IgM or both) and expressed cytoplasmic immunoglobulin. They were positive for the B-cell markers Leu 12 and Leu 16, and also for HLA-DR and the transferrin receptor. The cells lacked CD3 and the IL-2 receptor. Supernatants from these cell lines had autostimulatory activity. When 24-h culture supernatants were added to freshly cultured peripheral blood mononuclear cells, the proliferation index was 2-3 times higher as compared to cells cultured with HB101 medium alone. This autostimulatory activity can be attributed to a B-cell growth factor since these supernatants were also able (1) to support the growth of BD9 cells and (2) to augment the proliferation of PB B cells preactivated with S. aureus Cowan strain I. Furthermore, the supernatants did not contain IL-1, IL-2, or gamma-interferon. Thus, B cells that grow spontaneously from the peripheral blood of SS patients spontaneously produce a B-cell growth factor. This factor could contribute importantly to the autoantibody production, tissue lymphoid infiltration and B-cell lymphoma seen in this disease.

Estrogen induces normal murine CD5+ B cells to produce autoantibodies.

Females have better humoral immune responses and are more susceptible to autoimmune diseases than males. Normal female mice (C57BL/6J, C3H/HeJ, and NZW) have significantly increased spontaneous autoimmune plaque-forming cells (APFC) to mouse erythrocytes pretreated with bromelain (Br-ME) in spleen, peritoneal exudate cell, and bone marrow compared to their male counterparts. A minor subpopulation of B cells, CD5+ B, is thought to produce this autoantibody. As determined by dual color flow cytometry, increased APFC to Br-ME in females is not due to quantitative increase of CD5+ B cells. Rather, it is due to increased numbers or percentages) of CD5+ B cells producing these autoantibodies, because CD5+ B cells from females produced greater numbers of APFC to Br-ME than equal numbers of cells derived from males. The increased autoantibody production in females can be attributed to the effect of estrogen on the immune response because this hormone markedly augments APFC to Br-ME in intact or orchidectomized males. Male hormone had little effect. Importantly, estrogen did not increase the numbers of B or CD5+ B cells but augmented the ability of B cells to produce this response. This was verified when a T cell-depleted B cell fraction or fluorescence-activated cell sorter purified CD5+ B cells from estrogen-treated mice proved more efficient in the production of APFC to Br-ME. These results suggest that the number of CD5+ B cells committed to produce autoantibodies to Br-ME is increased under the influence of estrogen. This is the first demonstration that estrogen can augment the production of natural autoantibodies in normal mice. The overall augmented humoral immune responses in females and the B cell hyperactivity in female predominant autoimmune diseases appears to be due to estrogen.

The expression and function of CD3 and CD5 in patients with primary Sjögren's syndrome.

We studied the expression and function of 2 cell surface markers that induce T cell activation, CD3 and CD5, in 19 patients with primary Sjögren's syndrome (SS). The expression of CD3+ lymphocytes was normal, but CD3 function was moderately reduced, as measured by anti-CD3-induced T cell proliferation. Anti-CD3-induced stimulation of T cell help for Ig production and non-major histocompatibility complex-restricted (natural) killing were normal. In contrast, there was reduced expression and function of the CD5 molecule on peripheral blood lymphocytes of the SS patients. The ratio of CD5+ to CD3+ lymphocytes was 0.45 in SS patients compared with 0.85 in normal subjects, indicating that the CD3+ cells are relatively CD5-deficient in SS patients. Anti-CD5 monoclonal antibody did not augment suboptimal anti-CD3 stimulation of whole peripheral blood lymphocytes or of purified T lymphocytes from SS patients, which indicated impaired functioning of the CD5 molecule. A significant impairment in proliferation was found in response to phorbol myristate acetate and to ionomycin in combination, suggesting defective intracellular signaling. Findings from serial studies of individual patients suggested that normal or low expression of CD5 on T cells can be stable over periods as long as 2 years. However, in 2 patients who required systemic therapy, a correction of the CD5 lymphocyte abnormality occurred in association with clinical remission, which suggests the potential reversibility of this condition. Our findings suggest a possible defect in intracellular signaling in primary SS, related in part to the CD5 molecule, which may provide a molecular explanation for the T cell hyporesponsiveness that is characteristic of this disease.

Synergistic interaction between anti-CD3 and IL-2 demonstrated by proliferative response, interferon production, and non-MHC-restricted killing.

Anti-CD3 monoclonal antibody acts on normal peripheral blood mononuclear cells to induce T cell proliferation, interferon-gamma production, and non-MHC-restricted cytotoxicity against both NK (CD16+)-sensitive and -resistant target cells. Moreover, anti-CD3 and interleukin 2 (IL-2) act synergistically to give greater proliferative, interferon-gamma (IFN-gamma), and natural cytotoxicity responses than those expected by the simple addition of the individual responses to each stimulus acting alone. This synergistic response is macrophage independent, greatest at low concentrations of anti-CD3, inhibited by anti-IL2 receptor, and depends upon the induction of IL-2 receptors by CD3 activation which are then available to respond to exogenously added IL-2. Natural cytotoxicity induced by anti-CD3 and IL-2 correlates with IFN-gamma production, is inhibited by anti-IFN-gamma, and is still present after depletion of CD16-positive cells by specific monoclonal antibody and complement. The use of anti-CD3 in concert with IL-2 may be worthy of examination in a clinical setting, presumably because CD3/IL-2-generated LAK effector cells could be followed by in vivo administration of potentially lower and less toxic quantities of IL-2 than have been used in the past.

Defective induction of T-cell help and natural killing following anti-CD3 stimulation of autoimmune lymphocytes.

Anti-CD3 monoclonal antibody (MoAb) stimulates T cells in normal peripheral blood to proliferate and develop cytotoxic activity against NK-sensitive tumor cell lines. We now find that anti-CD3 MoAb also generates cytotoxic activity against a cell line (MEL-21) resistant to classical NK cell killing. After activation in vitro with anti-CD3 MoAb for 18 h, normal peripheral blood mononuclear cells (PBMNC) develop more HLA-DR-positive helper than suppressor T cells, manifest a functional helper effect as measured by increased IgG synthesis (P less than 0.01), as well as kill MEL-21 target cells. PBMNC from rheumatoid arthritis (RA) patients respond normally but mononuclear cells from rheumatoid arthritis synovial fluid (RASF) respond poorly. PBMNC from systemic lupus erythematosus (SLE) patients also respond poorly to anti-CD3 stimulation. Thus, the ability of anti-CD3 to stimulate IgG production and generate enhanced natural cytolytic activity are defective in both RASF and SLE lymphocytes.

B cells expressing CD5 are increased in Sjögren's syndrome.

In this investigation of B cells expressing the CD5 (Leu-1) cell surface marker, we found increased numbers of these cells in 13 of 19 patients with primary Sjögren's syndrome (SS) (68%), as well as in the rheumatoid arthritis patients. The percentage of B cells that demonstrated increased expression of CD5 was 46% in SS patients, 47% in rheumatoid arthritis patients, 24% in systemic lupus erythematosus patients, and 26% in normal subjects. Over a 2-year period, CD5 expression on B cells was a stable finding in several patients, except for 2 who required either steroid therapy or combined chemotherapy and irradiation for malignant lymphoma. Both of these patients had clinical remissions and their levels of CD5+ B cells returned to normal. The first patient had a clinical picture of SS/systemic lupus erythematosus overlap, associated with polyclonal B cell activation and decreased production of interleukin-2 in response to stimulation with phytohemagglutinin. These cellular immune abnormalities returned to normal after the institution of corticosteroids. Our observations suggest a relationship between the CD5+ B cell abnormality and disease activity. The results are discussed in relation to immunoregulatory properties of CD5+ B cells in autoimmune mice and the characteristic predisposition to malignant lymphoma among SS patients.