Symmetric division of cancer stem cells--a key mechanism in tumor growth that should be targeted in future therapeutic approaches.

As cancer stem cells (SCs) drive tumor growth, it is only through the elimination of those cancer SCs that a pharmacologic cure can be attained. To study ways to develop drugs that target cancer SC, we investigated changes in cellular mechanisms and kinetics that occur in SC populations during colorectal cancer (CRC) development. We used computer modeling to determine which changes could give rise to exponential increases in both SC and non-SC populations in CRC. Our results show that the only mechanism that can explain how these subpopulations increase exponentially in CRC development involves an increase in symmetric SC cell division. This finding suggests that any systemic therapies designed to effectively treat CRC and other cancers must act to control or eliminate symmetrical cancer SC division in tumors, while minimally affecting normal SC division in non-tumor tissues.

Computer modeling implicates stem cell overproduction in colon cancer initiation.

On the basis of our investigation of the premalignant crypt phenotype in familial adenomatous polyposis patients, the hypothesis is developed that tumor initiation in the colon is caused by crypt stem cell overproduction. A novel kinetic model for the colonic crypt was used to investigate how the earliest tissue abnormality (altered crypt labeling index) arises in these patients who have a mutant APC genotype. Only an increase in crypt stem cell number, not changes in the rate of cell cycle proliferation, differentiation, or apoptosis of the non-stem cell population, simulated this abnormality. This suggests that APC regulates the number of stem cells in the colonic crypt and when the cells become mutant, an expansion of the crypt stem cell population results.

A kinetic study of factor I-mediated modification and release of RBC-bound DNA-anti-DNA immune complexes.

A tritiated DNA-anti-DNA complement--containing immune complex probe was used to define the mechanism of factor I-mediated modification and release of erythrocyte CR1-bound immune complex in 10 normal control subjects. Experiments were performed with preformed immune complex, autologous erythrocytes, and autologous serum. Control experiments were performed by using a single donor source of erythrocytes and commercial factor I. The data demonstrate that the modification-release rate was first order with respect to factor I concentration, the mean half-life of red blood cell-bound immune complex was 1.7 +/- 0.7 minutes, and the mean percent of total immune complex present as unbound complexes was 19 +/- 11. These data are consistent with a clearance model in which factor I-mediated modification and release of red blood cell-bound immune complex plays a significant role in immune complex handling.

Mononuclear phagocyte system dysfunction in murine SLE: abnormal clearance kinetics precede clinical disease.

Although several studies have reported abnormal immune clearance in murine models of systemic lupus erythematosus (SLE), a consistent defect in mononuclear phagocyte function in SLE-prone mice has not been described. To evaluate the mechanism(s) of immune clearance in murine SLE, we applied the technique of kinetic analysis to clearance studies of radiolabeled, immunoglobulin-sensitized red blood cells in normal BALB/c and autoimmune BXSB, MRL-lpr/lpr, New Zealand black (NZB) and New Zealand black/white (NZB/W) mice. Clearance studies were performed in 4-week-old to 18-month-old mice with a complement-fixing rabbit IgG antimouse red blood cell antibody. Four clearance rate constants governing complement- and Fc-mediated clearance function were evaluated: complement-mediated sequestration (k1), C3b deactivation and release (k2), complement-dependent phagocytosis (k4), and Fc-mediated sequestration and phagocytosis (k3). BXSB male, MRL-lpr/lpr female and male, NZB female, and NZB/W female and male mice all had significantly decreased Fc-mediated clearance function (k3) when compared with control BALB/c mice (p less than 0.0001). This defect in Fc-mediated clearance was present in all four strains of autoimmune mice by 6 months of age and preceded the onset of serologic and clinical disease activity in NZB mice. Abnormal complement-mediated clearance was detected in MRL-lpr/lpr female and male mice, NZB female, and NZB/W female and male mice, but not in BXSB mice. In MRL-lpr/lpr mice decreased complement-mediated sequestration (k1, p less than 0.0001) and complement-dependent phagocytosis (k4, p less than 0.0001) were present as early as 4 weeks of age. In contrast, the change in complement-mediated clearance in NZB and NZB/W mice was characterized by decreased C3b deactivation and release (k2, p less than 0.001) and resulted in an enhanced early phase of clearance. Decreased k2 values in New Zealand mice occurred as early as 2 months of age, preceding serologic and clinical disease activity as well as decreased Fc receptor function. These studies demonstrated an early, progressive, and uniform defect in Fc-mediated clearance in the four murine strains of SLE studied. Complement-mediated clearance, however, varied considerably in lupus-prone mice, ranging from severe impairment in MRL-lpr/lpr to normal function in BXSB and accelerated clearance in NZB and NZB/W mice. Accelerated clearance in New Zealand mice was characterized by decreased C3b deactivation and release of antibody sensitized cells, which in turn led to increased phagocytosis of sensitized cells sequestered by complement-dependent processes.

Altered erythrocyte CR1 binding kinetics compensate for decreased binding capacity in rheumatoid arthritis.

Patients with rheumatoid arthritis have decreased numbers of CR1 per erythrocyte and decreased binding of immune complexes to erythrocytes. Overall erythrocyte immune complex binding activity depends on both the number and the binding kinetics of CR1. We measured kinetic parameters for the interaction between a complement-containing dsDNA:anti-dsDNA probe and erythrocytes in patients with rheumatoid arthritis and normal controls. The results indicate that: 1) the maximum quantity of immune complexes bound per erythrocyte was significantly decreased in rheumatoid arthritis compared with normal controls (p less than or equal to 0.009); 2) the steady state binding constant, Kss, and the association rate constant for binding of immune complexes to erythrocytes, ka, were significantly increased in rheumatoid arthritis versus normal controls (p less than or equal to 0.0001 and 0.002 respectively); 3) the dissociation rate constant for the release of bound immune complexes from erythrocytes, kd, was slightly smaller in rheumatoid arthritis but this difference was not statistically significant; and 4) the energies of activation for the association and dissociation reactions, Eaa, and Ead, did not differ between the two groups. These data confirm that while the maximum quantity of immune complexes bound per erythrocyte is decreased in rheumatoid arthritis, the association rate constants are larger and dissociation rate constants slightly smaller than those of normal controls. Changes in these kinetic parameters compensate for the decrease in the maximum quantity of immune complexes bound per erythrocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Mononuclear phagocyte system function in BALB/c mice: kinetic analysis of antibody-sensitized erythrocyte clearance in complement-depleted animals.

The mechanisms of immune clearance in normal BALB/c mice were studied by kinetic analysis of the clearance of immunoglobulin-sensitized red blood cells. A rate equation, derived from a model for the clearance of sensitized cells, was used to quantitate four rate constants regulating the individual rate-determining steps in the overall clearance process. A linear relationship was demonstrated between the level of antibody sensitization and constants regulating complement-dependent sequestration (k1), deactivation and release of cells back into the circulation (k2), complement-dependent phagocytosis (k4), and Fc-mediated sequestration and phagocytosis (k3). Clearance rate constants did not change with age in either female or male mice; nor was there a significant difference between the mean values in female versus male mice. The validity of the model was tested by altering serum complement levels to determine whether the predicted changes in complement-dependent rate constants k1, k2, and k4 would occur. Depletion of serum complement by cobra venom factor resulted in a significant decrease in complement-dependent sequestration (k1) and phagocytosis (k4) (p less than 0.001) but had no significant effect on Fc-mediated clearance function (k3). As serum complement was replenished, a greater-than-normal percentage of red blood cells sequestered by the complement clearance pathway underwent phagocytosis (k4) rather than being deactivated and released back into the circulation (k2). Demonstration that the derived rate constants are predictably sensitive to manipulation of a major clearance factor increases the confidence in using this technique and model to study immune clearance in experimental and naturally occurring disease states.

High-dose, pulse intravenous methylprednisolone enhances Fc gamma receptor-mediated mononuclear phagocyte function in systemic lupus erythematosus.

The benefit of high-dose, pulse intravenous methylprednisolone (IVMP) for some patients with active lupus nephritis would appear paradoxical, since active nephritis is associated with profound abnormalities in Fc gamma receptor function, and several studies have demonstrated that glucocorticoids decrease monocyte Fc gamma receptor expression and phagocytic function. To resolve this paradox, we investigated the possibility that pulse IVMP might enhance monocyte Fc gamma receptor function in patients with systemic lupus erythematosus (SLE). Circulating immune complex (CIC) levels, Fc gamma receptor-mediated clearance, and Fc gamma receptor-dependent monocyte function were analyzed in 23 SLE patients before and after pulse IVMP (1 gm daily for 3 days). A biphasic response in CIC levels, determined by a staphylococcal protein A binding assay, was observed. Initially, CIC levels increased within 2-4 hours after the first dose of pulse IVMP and then decreased by 50% within 24-48 hours after the completion of therapy. Fc gamma receptor-mediated binding and phagocytosis of IgG-sensitized erythrocytes (EA) by monocytes in vitro were significantly enhanced 24 hours after the final dose of pulse IVMP (pre-IVMP versus post-IVMP 43 +/- 14% versus 53 +/- 12% EA rosettes, P less than 0.01; 3.00 +/- 1.04 versus 3.99 +/- 1.30 EA ingested/monocyte, P less than 0.01). In contrast, there was no change in the phagocytosis of an Fc gamma receptor-independent probe, neuraminidase-treated erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

A kinetic study of erythrocyte-DNA/anti-DNA immune complex association and dissociation reactions in systemic lupus erythematosus.

Decreased binding capacity of the erythrocyte complement receptor (RBC CR1) in systemic lupus erythematosus (SLE) may contribute to abnormal handling of circulating immune complexes in these patients. Decreased numbers of RBC CR1 have been reported in SLE, but, since binding is a function of both receptor number and receptor binding kinetics, we measured kinetic parameters for the interaction of complement (C) containing [3H]DNA:anti-DNA immune complexes (IC) with normal control (NC) and SLE RBC. Experiments were performed at five temperatures ranging from 7-37 degrees C. The parameters measured included: (1) the maximum quantity of DNA:anti-DNA:C which could bind per RBC, S; (2) the association rate constant, ka, for the binding of DNA:anti-DNA:C to RBC; (3) the dissociation rate constant, kd, for the dissociation of bound DNA:anti-DNA:C IC from RBC; (4) the steady-state constant, Kss (ka/kd); and (5) the energies of activation for association, Eaa, and dissociation, Ead. Although the relative amount of bound DNA:anti-DNA:C per RBC was significantly decreased in SLE patients compared to NC (P less than 0.001), the mean values for Kss, ka, kd, Eaa and Ead did not differ significantly between the two groups. These data suggest the following: (1) RBC CR1 binding and dissociation of DNA:anti-DNA:C are consecutive reactions resulting in steady-state concentrations of free and RBC-bound IC; (2) at steady-state times, the ratio of RBC bound to unbound DNA:anti-DNA:C are governed by kinetic factors; (3) since the binding kinetics of SLE and NC RBC are not significantly different, the decreased binding activity described by other investigators can only be due to a decreased number of CR1 per RBC; and (4) values for Eaa and Ead suggest that the rate-determining steps in IC association with and dissociation from RBC involves making and breaking of hydrogen bonds.

Mononuclear phagocyte system complement receptor dysfunction in rheumatoid arthritis.

To explore the relationship between complement-dependent and Fc receptor-mediated clearance mechanisms, clearance studies were performed in nine patients with definite rheumatoid arthritis and 49 normal controls. Kinetic analysis allowed evaluation of the four rate constants governing both complement- and Fc-mediated clearance processes. This analysis revealed that patients with rheumatoid arthritis had significantly reduced values for the constants regulating complement-dependent clearance (p less than 0.001). Complement-mediated clearance dysfunction was associated with normal serum complement levels and normal Fc receptor function. These data indicate that complement-mediated clearance defects are neither the simple result of a hypocomplementemic complement opsonization deficiency nor merely the reflection of profound Fc dysfunction. Defects in the two clearance processes can occur independently. These data demonstrate a specific complement receptor defect in the fixed tissue macrophages of patients with rheumatoid arthritis.

Mononuclear phagocyte system in SLE. II. A kinetic model of immune complex handling in systemic lupus erythematosus.

Using the principles of reaction kinetics, we constructed a model for the handling of immune complexes and the pathogenesis of SLE immune complex disease. The model incorporates rate constants for complement- and Fc-mediated clearance, parameters for autoantibody, complement and immune complex levels, and scores for clinical disease activity. The model assumes that complement fixation by immune complexes is a prerequisite for complement-mediated clearance and that disease activity results from immune complex deposition. To test the relationships derived, data from 32 lupus patients were analyzed and the predictions were compared with actual findings. The model predicts a low correlation coefficient between disease activity and immune complex levels (found, r = 0.25, p greater than 0.1). The model also predicts a poor correlation between disease activity and impaired Fc-mediated clearance in patients with normal complement levels (found, r = 0.10, p greater than 0.1), but a high correlation coefficient between disease activity and impaired Fc-mediated clearance in patients with hypocomplementemia (found, r = 0.61, p less than 0.001). In patients with normal complement levels, the model predicts a good correlation between anti-DNA antibody and immune complex levels (found, r = 0.71, p less than 0.001), whereas hypocomplementemic patients should have a good correlation between anti-DNA to CH50 ratios and immune complex levels (found, r = 0.73, p less than 0.001). The model predicts that disease activity should correlate better with the product of the anti-DNA to CH50 ratio and the rate constant for Fc-mediated clearance than with any single parameter (found, r = 0.85, p less than 0.0001). These significant correlations, which were predicted by the model, suggest that complement-mediated mechanisms are the first line of host defense against immune complex-induced injury, that the efficiency of complement opsonization plays a central role, and that both abnormal complement- and Fc-receptor function leads to active renal disease in SLE.

Mononuclear phagocyte function in SLE. I. Bipartite Fc- and complement-dependent dysfunction.

To determine the relative contributions of Fc- and complement-mediated immune clearance to the overall mononuclear phagocyte system (MPS) dysfunction in SLE, we performed a kinetic analysis of clearance rate data from 32 patients and 49 normal controls. Three rate constants regulating complement-mediated MPS clearance and one rate constant regulating Fc-mediated MPS clearance were evaluated. Mean values for rate constants regulating complement-mediated phagocytosis (k4) and Fc-mediated clearance (k3) were significantly lower for the patient population as a whole when compared with normal controls (p less than 0.01 and p less than 0.001, respectively). Further analysis by clinical subgroups revealed that mean k3 values were significantly low for all but the inactive nonrenal subset of patients, whereas mean k4 values were significantly low for both the active and inactive renal patients but not for nonrenal patients. Rate constant values for Fc-mediated clearance correlated significantly with disease activity scores for the entire SLE group (p less than 0.001) as well as for the subset of patients with active and inactive renal disease (p less than 0.001). Neither k3 nor k4 correlated significantly with anti-DNA antibody, titers, total hemolytic complement levels, or circulating immune complexes. These data indicate that both Fc- and complement-mediated clearance defects occur in SLE. Nonrenal patients have at least one clearance mechanism intact, whereas immune complex glomerulonephritis is associated with dysfunctions in both of these clearance mechanisms.

The quantitative relationship between clearance rate constants and number of C1-fixing sites.

The rate equation correlating kinetic data for immune-mediated clearance of RBC in guinea pigs was extended to human studies. Rate constants were evaluated for clearance of IgG, IgM, and cold agglutinin-sensitized RBC in normal human volunteers as well as for clearance of IgM-sensitized cells from a small group of patients with primary biliary cirrhosis. All rate constants evaluated in the clearance of sensitized RBC were found to correlate with the number of C1-fixing sites. These correlations not only provide additional support for the mechanism from which the rate of equation was derived, but also allow a quantitative prediction of rate constants and clearance patterns based on the level of sensitization. In addition, the analysis of data from abnormal clearance studies produced rate constants that provided both identification and quantification of the specific defective reaction(s).

A kinetic analysis of immune-mediated clearance of erythrocytes.

A mathematical expression has been derived that successfully correlates the kinetic data for the immune-mediated clearance of red blood cells. The expression resulted from the solution of differential equations arising from a clearance mechanism that was, essentially, consistent with that described by Schreiber and Frank. The mathematical expression correlated data for both IgG-and IgM-mediated reactions. Four different rate constants appear in the final kinetic equation; these constants, which measure the rates of the various steps in the clearance process, were evaluated by an iterative curve-matching process. The values of the rate constants were found to be dependent upon type of sensitizing immunoglobulin, number of C1-fixing sites, and several known immune system modifiers. Correlation of the derived rate expression with the experimental data provided a critical test for the Schreiber-Frank mechanism and the values of the rate constants provided additional insights into the immune clearance process.