Proc. Natl. Acad. Sci. USA

Vol. 76, No. 6, pp. 2984-2987, June 1979 Medical Sciences

Virolysis of mouse mammary tumor virus by sera from breast cancer patients (virolytic assay/reverse transcriptase/human cancer/retrovirus)

STEVEN S. WITKIN, RUDI A. EGELI, NURUL H. SARKAR, ROBERT A. GOOD, AND NOORBIBI K. DAY Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Contributed by Robert A. Good, March 30, 1979

All type C retroviruses are lysed by human ABSTRACT serum in apparently antibody-independent, complement-mediated reactions. In contrast, we have now determined that the mouse mammary tumor virus (MMTV), a type B retrovirus, is not disrupted by normal human serum. MMTV was lysed, however, when rabbit antibody to whole MMTV was added to the serum. By taking advantage of this dependence of MMTV lysis on specific antibody, a virolytic assay was developed, based on the measurement of reverse transcriptase released from disrupted virions, to search for evidence of antibodies to MMTV in human sera. Significantly greater virolytic activity was detected in the sera of patients with breast cancer than in sera of patients with benign disease (P < 0.001) or colorectal cancer (P < 0.001) or in sera from apparently healthy individuals (P < 0.002). This assay thus appears to be able to detect a unique attribute, possibly the presence of an antibody crossreacting with MMTV, in serum of patients with breast cancer. Antigens related to mouse mammary tumor virus (MMTV) proteins appear during the development of breast cancer in both mice and humans. In mice with mammary cancer, plasma levels of the MMTV coat protein (gp52) are increased (1). In addition, MMTV-producing mouse cells grown in vitro have been shown to shed gp52 and its precursor into the culture medium (2). Studies in Spiegelman's laboratory have demonstrated that human breast cancer tissue contains an antigen immunologically related to MMTV gp52 (3) as well as particles encapsulating an MMTV-like reverse transcriptase (4). Brief reports that sera from breast cancer patients, but not from normals, contain antibodies reactive with MMTV have appeared (5, 6). Black et al. (7) have shown that leukocytes of breast cancer patients were responsive, by a leukocyte migration procedure, to MMTV-containing mouse milk and to purified MMTV. These MMTV-responsive leukocytes were also responsive to pathological tissues of patients with in situ breast cancer. In contrast, invasive breast cancer tissue did not evoke a significant lymphocyte response. It thus appeared that the antigenicity of breast cancer tissue was attributable to a component related to MMTV only at the earliest stages of development of the cancer. In this communication, we report the development of a virolytic assay to detect antibodies to MMTV. When applied to human serum, a significant difference (P < 0.001) was found in MMTV lytic activity between sera of breast cancer patients and all other human sera tested.

as described (9). The type C baboon endogenous virus (BEV), purified from culture supernatants of infected baboon kidney-canine thymus (BKCT) cells, was generously provided by Sol Spiegelman (Columbia University Institute of Cancer Research). Buoyant density centrifugation of the viruses in 15-60% (wt/wt) sucrose gradients was as described (10). Blood samples were obtained from previously untreated patients on admission at our hospital as well as from healthy, age-matched volunteers. The blood was allowed to clot for 1 hr at room temperature, clarified by centrifugation, divided in small portions, and stored at -70°C. Virolytic Assay. Aliquots (25 p1) of each serum to be tested were mixed with MMTV, BEV, or 0.01 M Tris-HCl (final volume, 50 ,ul) and incubated at 37°C for 30 min. Reaction components for the measurement of released reverse transcriptase were then added (see below) for the assay of virolytic activity. Equal amounts of virus incubated in 0.01 M Tris-HCl at pH 7.2 or in 0.01 M Tris-HCl plus 0.05% Triton X-100 were always assayed along with our samples to obtain values for 0 and 100% lysis, respectively. Viruses incubated in buffer always exhibited less than 5% lysis; these percentages have been subtracted from the serum-derived values. Reverse Transcriptase Assays. Reverse transcriptase assays for BEV (11) or MMTV (12) were by published procedures. The samples contained, in a total volume of 100 ,ul: 0.5 ,ug of (dT)12 18poly(rA) (P-L Biochemicals, Milwaukee, WI), 0.1 ,umol of dithiothreitol, 5 ,umol of Tris-HCI at pH 7.8, 0.5 ,umol of MgCl2 (with MMTV) or 0.06 ,umol of MnCl2 (with BEV), and 5 ,uCi of [methyl-3 H]dTTP (17.8 Ci/mmol, 1.6 X 104 cpm/ pmol; 1 Ci = 3.7 X 1010 becquerels) (New England Nuclear). Incubation was at 37°C for 60 min. Reactions were terminated by the addition of 2 ml of cold 5% trichloroacetic acid/1% Na pyrophosphate. Acid-insoluble precipitates were collected on glass fiber filters (type A/E, Gelman Instrument Co., Ann Arbor, MI) and radioactivity was determined (13). Radioactivity measured in serum samples incubated without virus (200-300 cpm) was subtracted from experimental values. All samples were counted to a standard error of ±2.5%; counting efficiency was 42%. Statistical Analysis. Distributions of lytic activity between different groups of sera were compared by means of Wilcoxon two-sample rank sum test; all results pertain to two-sided tests.

RESULTS Viral Lysis by Normal Sera. The disruption of all type C retroviruses by sera from apparently healthy humans has been demonstrated (14, 15). Viral lysis did not require specific antibody; instead it involved the direct activation of the classical complement pathway (16) by a protein on the viral surface (17).

MATERIAL AND METHODS Viruses and Sera. MMTV was purified from the culture supernatant of the MMTV-infected epithelial cell line MuMT-73 (8). Rabbit antiserum to whole MMTV was prepared The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: MMTV, mouse mammary tumor virus; BEV, baboon endogenous virus; gp52, 52,000-dalton glycoprotein. 2984

Proc. Natl. Acad. Sci. USA 76 (1979)

Medical Sciences: Witkin et al.

2985

0

1.0

'E I [ L1 *-4 ° V

~S

,800

1.3

800

p

1.24

p

e ru m

1.19

0~~~~~~~~0 1.2

600

400

1.1

400

200

1.0 200

O

600

1.2

1.0

20 10 0 20 10 Fraction Fraction FIG. 1. Buoyant density of BEV (Left) and MMTV (Right) after incubation with buffer (Upper) or human serum (Lower). Virus (2.5 Aig) was added to 0.01 M Tris-HCl or pooled human serum (final volume, 50 Al) and incubated at 370C for 30 min. The samples were then layered on top of 5 ml of 15-60% (wt/wt) sucrose gradients in phosphate-buffered saline (13) and centrifuged in a swinging bucket rotor for 16 hr at 195,000 X g. The gradients were fractionated, density (O ---0) was determined from refractive indices and aliquots were assayed for reverse transcriptase

0

activity (0-0).

It was of interest to ascertain if MMTV, a type B retrovirus, was similarly affected. Various concentrations of MMTV or BEV were added to 25 pi of pooled normal human serum (final volume, 50 Al) or 0.01 M Tris-HCl with or without 0.05% Triton X-100 and incubated at 370C for 30 min. The samples were then placed in an ice-water bath and 50 ,ul of the [3H]dTTPcontaining reverse transcriptase assay mixture was added. After an additional incubation at 370C for 60 min, acid-insoluble radioactivity in the samples was determined. Over the range 0.1-1.0 ,ug, BEV was completely lysed by human serum but less than 10% of MMTV was lysed; neither virus was lysed by 0.01 M Tris-HCI (Table 1). The effect of individual normal human sera on viral disruption is shown in Table 2. Each of the human sera, but not Table 1. Effect of human serum on the lysis of MMTV or BEV %o lysis* Concentration, Serum Buffer Virus ug MMTV

BEV

0.1 0.2 0.5 1.0

0 0 1 0.5

9 6 8 8

0.1 2 108 2 0.2 90 103 0.5 0 2 91 1.0 * Lysis was measured by the release of viral reverse transcriptase. The percentages were computed by using the reverse transcriptase activity released from the same concentration of virus treated with 0.05% Triton X-100 as 100%. For 1 Mg of virus, 100% lysis = 3.2 pmol (for MMTV) or 3.0 pmol (for BEV) of [:3H~dTTP polymerized.

rabbit sera, elicited the disruption of BEV but none was effective against MMTV.

The effect of human serum on viral integrity was further examined by an experiment in which 2.5 ,yg of BEV or MMTV was incubated in either 0.01 M Tris-HCl or pooled human serum, as described above, and then subjected to equilibrium centrifugation in 15-60% sucrose gradients. The gradients were fractionated, densities were determined from refractive indices, and aliquots of each fraction were assayed for reverse transcriptase activity in the presence of 0.5% Triton X-100. The BEV preparation incubated in buffer yielded two peaks of reverse transcriptase activity, at densities corresponding to intact virus (1.17 g/ml) and viral cores (1.21 g/ml). By contrast, with BEV incubated in serum, reverse transcriptase activity was primarily located at the top of the gradient (Fig. 1 left), confirming viral lysis. Although the MMTV preparation incubated in buffer also yielded activity at viral (1.19 g/ml) and viral core (1.24 g/ml) densities, the effect of serum on MMTV was totally unlike its effect on BEV. In this case (Fig. 1 right), the banding pattern of MMTV in serum was the same as it was in buffer. Table 2. Lysis of MMTV and BEV by various sera % lysis* BEV MMTV Serum Human A Human B Human C

0 1 3 4 2

101

106 88

2 Rabbit 90 Human A + rabbit * % lysis was calculated as in Table 1; 100% lysis = 1.2 pmol (for BEV) or 1.4 pmol (for MMTV) of [l3H]dTTP polymerized. Viruses were added at a concentration of 1 gg per reaction.

Medical Sciences: Witkin et al.

2986

Proc. Natl. Acad. Sci. USA 76 (1979) Table 3. Virolysis of MMTV by patient's sera No. of patients Breast Benign breast Colorectal Normal cancer % viral lysis* cancer disease

2000 2 El11500-

0-5 6-8

0

u

1000 [

5001 1:10

1:10O

1:1000

Antibody dilution - FIG. 2. Release of reverse transcriptase from MMTV by viral antibody plus human serum. Serial dilutions, in 0.01 M Tris-HCl of rabbit antiserum to whole MMTV were incubated in duplicate with 25 ul of pooled human serum and 1 ,ug of MMTV (final volume, 50 ,ul) for 30 min at 37°C. An equal volume of the [3H]dTTP-containing reverse transcriptase assay mixture was then added, and acid-insoluble radioactivity was determined after a 60-min incubation at 37°C; 100% lysis = 6254 cpm (0.4 pmol). Duplicate values differed by