immunohistochemical localization of a choriogonadotropin ... 1978... · immunohistochemical...

IMMUNOHISTOCHEMICAL LOCALIZATION O F A CHORIOGONADOTROPIN-LIKE PROTEIN IN

BACTERIA ISOLATED FROM CANCER PATIENTS HERNAN F. ACEVEDO, PHD, MALCOLM SLIFKIN, PHD

GAIL R. POUCHET, BA, AND MATIAS PARDO, BA

By the use of specific antibody to human chorionic gonadotropin (CG) as well as to its P-subunit, and the application of the indirect fluorescein-labeled and peroxidase-labeled antibody techniques, we have demonstrated the presence of a membrane (wall)-associated CG-similar immunoreactive protein in 15 strains of bacteria isolated from tissues of patients bearing malignant neoplasms. These microorganisms were classified as S. epzdermidis, (12), E. coli (2), and a single strain ofP. maltophilia (ATCC 13637). The absence of the CG-like antigen in other ‘‘cancer associated bacteria”, Streptococcus faecalis (ATCC 12818) and Pseudomonas aeruginosa (from patient with cancer of colon), demonstrated that not every ‘‘cancer associated bacteria” has the capability to synthesize the trophoblastic-like protein. The negative results obtained with a number of “noncancer control” bacteria of known origin, obtained from ATCC and from clinical samples, strongly supported the idea that the existence of these CG-like protein producing microorganisms is not a ubiquitous finding. The demonstration of a de nouo bacterial biosynthesis of a protein having similar antigenic and biophysi- cal properties to those of the human trophoblastic hormone, has great biological implications, especially if its biosynthesis is proven only in bacterial strains growing in the presence of cancer cells in which we have already demonstrated the presence of a similar antigen. The explanation of the phenomenon is unknown. Because of their origin, the potential of “genetic exchange” with subsequent expression of the mammalian gene by the bacterial cells becomes a possibility. It is also possible that the gene coding for the CG-like protein is normally present but inactive or repressed in all bacteria.

Cancer 4 1 : 12 1 7-1 229, 1978.

ECINNING IN 1948, VIRGINIA (WUERTHELE- B Caspe) Livingston and her associates have published a series of papers describing a pleo- morphic, acid-fast bacterium which she observed in, and isolated from, cancer tissues and body fluids of patients with malignant neo-

From the Division of Experimental Pathology and Section of Microbiology and Immunology, Department of Labora- tory Medicine, William H. Singer Memorial Research Insti- tute, Allegheny General Hospital, Pittsburgh, Pennsylvania.

The authors thank to Drs. Virginia Livingston, The Liv- ingston Clinic, Herman Cohen and Alice Strampp, Prince- ton Laboratories, Inc., and Lewis F. Affronti, George Wash- ington University Medical Center, for making available to us their strains of “cancer associated bacteria,” and Dr. Ver- non C. Stevens, The Ohio State University College of Medi- cine, and Mr. David R. Crockford, Serono Laboratories, Inc., for supplying us with some of the antiserum.

Address for reprints: H. F. Acevedo, Ph.D., Department of Laboratory Medicine, 320 East North Avenue, Pitts- burgh, Pennsylvania 15212.

Accepted for publication December 2, 1977.

plasms. In 1970, she and Eleanor Alexander- jackso on published a full description of the organism(s) and proposed the name Progenitor cryptocides as a new taxon within the Actinomycetales. They reported that the P. cryptocides cross-reacts antigenically with M . tuberculosis, with which it shares the property of acid fastness. 29

Based on the proven presence of a human chorionic gonadotropin (choriogonadotropin, CG)-like protein in the serum of some patients with different types of cancer, 13,14,21-23,34,37,40,48

and on her views concerning the nature of cancer, primarily in respect to her consideration of P. cryptocides as a possible etiologic agent of the disease, Livingston and her associates tested their bacterial isolates for the production of the trophoblastic-like protein. Their investigations demonstrated that these bacteria, when cultured in vitro produced a substance immunologically similar to the human trophoblastic hormone. 30

In 1976, Cohen and Strampp17 reported that 0008-543X-78-0400-1217-0120 @ American Cancer Society

1217

1218 CANCER

TABLE 1. “Control bacteria,” from American Type Culture Collection

1. Aeromonas hydrophila iubsp. formicans 2 . ( h t r i d i u m haemolilicum 3. (.%oslridium nougi Type A 4. Eschrrichiu coli 5. l~urohacterii~m riucleatum 6. Huemophilus aqyp tus 7. I,aclobardlur casei 8. .Iljcoplamia huminis Type 1 9. .Ilr/raxrlle lacunata

10. I’reudomonas aeruginosa 11. PrriLrionionaJ mallophilta

12. l’seudomonar lrrlosteroni 13. Serratia rriarcrsrens 14. Stafihylocriccus epidermtdi.s 1.5, Strr~~tobecillirs monill/ormis 16. ,Struptococcur mirtans

ATCC 13 137 ATCC 9650 ATCC 19402 ATCC 25922 ATCC 25586 ATCC 11116 ATCC 7469 ATCC 23114 ATCC 17067 ATCC 27853 ATCC 13270 ATCC 13636 ATCC 17448 ATCC 17666 ATCC 11 996 ATCC 13880 ATCC 14970 ATCC 14647 ATCC 2.5175

they had obtained two cultures of P. cryptocides from Dr. Livingston and confirmed the production of a CG-like glycoprotein not only by radioimmunoassay (KIA) using specific antibody to the P-subunit of the trophoblastic but also by two assays for specific biologic activity, the radioreceptor analysis41 and the testosterone stimulating assay. l9 The glycoprotein nature of the material was demonstrated by its chromatographic behavior on Concanavalin A-Sepharose columns. ‘O

Cohen and Strampp stated in their publication that the bacteria received from Dr. Liv- ingston were Gram positive cocci. These investigators also reported that they isolated bacteria from the urine of a patient with terminal carcinoma of the colon which also produced CG-like material. In contrast to the material obtained from Dr. Livingston, these bacteria were Gram negative rods, identified as Escherichia coli by fermentation criteria.

Working independently, Affronti and his associates have also described the isolation of two CG-like protein producing microorganisms. One was classified as a Staphylococcus epidermidis strain obtained from patients with advanced carcinoma of the breast, and the other was an E. colz strain isolated from patients with adenocarcinoma of the C O I O ~ . ~ - ~

Because of our findings of the de noun biosynthesis of a CG-like glycoprotein by every cell classified as a cancer cell thus far tested, in tissue culture, in the experimental animal and in the human being, 1.2,42 we decided to include these bacteria in our ongoing investigations in order to study, by the specific immunohisto-

April 1978 Vol. 41

chemical techniques we are employing, the production and localization of the antigen, and by electron microscopy, their ultrastructural characteristics.

While the testing for the presence of the antigen in the cancer cells as well as in all the aforementioned bacteria was being carried out, Dr. Robert J. Ryan at the Mayo Medical School, in collaboration with Dr. Nancy D. Richert, isolated from the ovarian fluid of a sow two Gram negative, motile bacteria, classified as Pseudomonas maltophilia and Enterobacter cloacae on the basis of physiological, nutritional and biochemical characteristics. 39 Subsequent out- growth of the isolated strains in liquid media demonstrated that this porcine P. maltophilia was capable of binding ‘251-labeled hCG, whereas E. cloacae showed no gonadotropin binding. Among several strains of different bacteria utilized as controls, the investigators used a P. maltophilia obtained from American Type Culture Collec- tion (ATCC 13637). Their preliminary data showed that the P. maltophilia culture media con- tained a protein molecule crossreactive with antisera to total CG as well as to its P-subunit.

But the most important aspect concerning ATCC 13637 strain of P. maltophilia was that Richert and Ryan did not disclose in their publication the fact that this bacterium was originally isolated from the oropharyngeal region of a patient with mouth cancer. 25

Because of all these findings, a systematic in- vestigation for the presence of the CG-like antigen in bacteria from proven sources (known origin) was initiated in order to demonstrate if the CG-like antigen is ubiquitous in nature, or is present only in bacteria isolated from humans and animals with cancer, spontaneously devel- oped and/or experimentally induced. The preliminary results of our investigations are hereby reported.

MATERIALS AND METHODS “Control Bacteria” and “Cancer Associated Bacteria”

The “control bacteria” utilized in this work has been obtained from different sources. First, from American Type Culture Collection (ATCC), using strains which origin has been established with complete assurance (Table l ) , and second, from clinical specimens from patients in which the presence of cancer was ruled out with almost absolute certainty (Table 2). Young males, aged 8 to 18 years were the best source, since malignant lymphomas, brain neo-

N o 4 CG-LIKE PROTEIN IN BACTERIA Acevedo et al . 1219

plasms and testicular neoplasms, the most common malignancies associated with this age group, can be easily ruled out.

Nine samples of “cancer associated bacteria” were obtained from Dr. Virginia (W-C) Liv- ingston, San Diego, California. One of the cultures was obtained from a neoplastic tissue, and the remaining were isolated from urine samples. All the patients were very advanced cases repre- senting carcinomas, sarcomas and lymphomas. Six additional cultures were obtained from Dr. Lewis F. Affronti, George Washington Univer- sity Medical Center, Washington, D.C. All these samples were isolated from cancer tissue, three of them from patients with metastatic carcinoma of the breast and the other three from patients with carcinoma of the colon. The procedures for the primary isolation of the bacteria were those previously described by Livingston and Alexander-Jacksonm and by Diller and Do- nelly. l’

We also obtained from ATCC, strain 13637 of P. maltophilia, described as having been isolated from a patient with mouth cancer, and an additional bacterium, Streptococcus faecalis (ATCC 12818) which according to the ATCC Catalog was originally isolated from a patient with carcinoma of the gingival area.’

All bacteria were classified by standard bacte- riological procedures, that is by morphological, physiological, nutritional and biochemical characteristics, 49 including gas chromatographic procedures as described by Holdeman and Moore. 24 Gram staining and the Kynyoun acid- fast procedure was performed in all isolates. Biotyping of S. epidermidis was also performed according to the method of Baird-Parker. “Anti- biograms were done by the Bauer-Kirby method. l2 All cultures received from the different sources were stored at 4 C on Trypticase soy agar (BBL) and maintained in Trypticase soy broth containing 15% v/v glycerol at -70 C (43).

Cell Preparation The two immunohistochemical reactions, the

indirect fluorescein-labeled and the indirect peroxidase-labeled reactions, were carried out in unfixed air dried cells. The use of unfixed samples is of importance, since the cells maintain their morphology as well as their antigenicity.

Reagents Highly purified and specific antibodies are

obtained from different sources. This has the advantage of providing a way for monitoring

TABLE 2. “Control bacteria.” laboratory isolates

1. Acinefobacfer anztralus 2 . Aeromonas hydrophila 3. Uordetella bronchisepf ica 4 .Veisseria gonorrhoeae (10 strains) 5 . I’seudomonads:

a ) P. aeruginosa h ) P. cepacia c) P.,Juorescens d ) P. maltophilia e) P. putrefacten5 f ) P. Jtutzeri

a ) S. aureus (2 strains) b) S. epidermzdis (8 strains)

6 . Staphylococcus:

quality control and reproducibility, since samples of the same type of cells can be tested with antisera from different sources.

Specific rabbit antiserum to p-CG was supplied by Dr. Vernon C. Stevens, The Ohio State University College of Medicine, and by Serono Laboratories, Inc., Boston, Massachusetts. Spe- cific rabbit antiserum to total choriogonadotropin was supplied by the same sources, and was also obtained from Cappel Laboratories, Down- ingtown, Pennsylvania, and from Miles Labora- tories, Elkhardt, Indiana. All these antisera are utilized as first antibodies. Rabbit antihorse antiserum utilized for control, replacing the first antibody was obtained from Cappel Laborato- ries.

Fluorescein-labeled goat antirabbit antiserum was obtained from Cappel Laboratories and from Behring Diagnostics, Rochester, New York. Peroxidase-labeled goat antirabbit antiserum and the peroxidase-antiperoxidase complex were obtained from Cappel Laboratories. All these antisera are utilized as second antibodies. All antibodies were purified according to actual standard procedures at their point of origin.

T h e indirect immunoperoxidase reaction requires 3,3’-diaminobenzidine-tetrachloride (DAB) as peroxidase stain, 3.5 mg in 10 ml of phosphate buffered saline (PBS) with 0.015 ml of 30% hydrogen peroxide. Light Green (Yel- lowish SP) in 0.2% absolute methanol is used as counterstain, and Permount as mounting medium. Ninety-five percent glycerine in PBS, p H 7.0 f 0.2, 0.01 M is used as mounting media for the indirect immuno-fluorescein reaction.

Reagent Controls: Utilization of CG-absorbed first antibody (rabbit anti-serum to 0-CG and to total CG) and elimination and/or replacement of the first antibody by rabbit serum, rabbit

1220 CANCER April 1978 Vol. 41

were subsequently fixed in denatured alcohol for 15 minutes, washed in PBS for S-10 minutes, and stained with freshly prepared DAB for 10 minutes. PBS washings were repeated again followed by a one minute rinse in distilled water. Dehydration was then performed by washing twice for one minute in 95% alcohol. The samples were then counterstained with Light Green for 15-20 seconds (3 slow dips), destained in absolute methanol for 10-15 seconds (2 slow dips) and cleared with xylene. With the xylene still on the slide, permount and coverslip were then applied. The presence of the trophoblastic- like protein is indicated by a brown to almost black granular or continuous staining depend- ing on the concentration of the antigen.

The trophoblastic-like protein(s) is considered to be present only when the indirect fluorescein- labeled and the indirect peroxidase-labeled immunohistochemical reactions are positive by light microscope. This is essential because equivocal reactions in the immunoperoxidase reagent controls can sometimes be observed in some cells, possibly due to intrinsic peroxidase content.

Nonspecific fluorescence was not a problem since it is easily recognized by an experienced observer, and can be eliminated by the use of appropriate filters and/or counterstaining.

Instruments

The optical system for examining the preparations subjected to the indirect immunofluorescein reaction consists of a Leitz-Ortholux microscope fitted with a darkfield condenser, 10 Xocular and achromatic oil immersion objective (95 X; NA l.O), and a lamp housing with an Osram HBO 200 mercury vapor bulb. A BG-12 excitor filter is used in combination with a bar- rier filter No. 47. Photographs are made with a 35 mm Leitz camera using Eastman Kodak Photomicrography color film with exposure times from 40 to 60 seconds.

Transmission electron microscopy studies were performed using Philips 300 electron mi- croscopes, according to the method described by Slifkin et a1.“ in order to study the ultrastructural characteristics of the CG-producing bacteria in comparison to those of the “non- producer^".

antihorse antiserum and/or PBS were used as reagent controls.

In addition to the aforementioned standard controls for immunohisto-chemistry we also em- ployed, as an additional test for specificity, what we have called “cell controls,” which were proven CG-producing cells utilized as “positive controls.” These were the nonclonal human choriocarcinoma BeWo cell line given to us by Dr. Roland A. Patillo, from the Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin, 38 and a sub- line obtained from ATCC, strain CCL 98.

At the same time, proven “non-CG-producing” cells were utilized as “negative controls. ” An example of such “negative cell control” is the MA-169, a tissue cultured clone of human ade- nomatous prostatic hyperplasia, obtained from Microbiological Associates. In every instance, reagents giving a positive reaction with the “positive cell control” have given a negative reaction with “negative cell controls”.

Immunohistochemical Methods

The specimens were labeled following previously described techniques. 1,10*28,32,33938,42 F or the indirect immunofluorescein reaction, one or more drops of the first antibody were added in order to cover all the preparation on the slide. The samples were then incubated at room temperature in moist atmosphere for 30 minutes, using a moist ambient temperature incubator. The slides were then gently agitated on a Clini- cal Rotator (Eherbach Corporation, Ann Arbor, Michigan) for a period of 10 minutes. After completion of this time, the slides were removed and drained briefly. Without allowing the preparation to dry, each sample was then treated with one or more drops of fluorescein-labeled second antibody. The 30 minute incubation was repeated, followed by rinses and washes as above. The slides were then drained and air dried. A drop of BACTO mounting fluid (Difco Laboratories, Detroit, Michigan) and coverslip were applied. Samples were then ready for ex- amination with the ultraviolet microscope. When present, the CG-like antigen is recognized by an apple green fluorescence at the foci of the protein. From the moment of reconstitution of the fluorescein-labeled second antibody, all work must be done in a darkened laboratory.

The procedure followed for the indirect immunoperoxidase reaction was identical to the method used for the indirect immunofluorescein reaction up to the rinses and washes done following the incubation with the second antibody, this time peroxidase-labeled. The preparations

RESULTS

Figures 1 and 2 illustrate the results of the immunohistochemical reaction as applied to the “positive cell control” and to the “negative cell control” respectively. These “cell controls”

No. 4 CG-LIKE PROTEIN IN BACTERIA Acevedo et al. 1221

of the great production of the antigen, CG, nonspecific attachment of the fluorescein-tagged second antibody molecules to the cell did not occur.

It is necessary to stress here that all the aforementioned controls with both markers as well as with “control cells” are performed every time cell tests are done, and every time new reagents are received.

With respect to the “cancer associated bacteria,” all the cultures received from Dr. Liv- ingston as well as 2 of the 3 isolated from breast carcinoma patients by Dr. Affronti were classified as Staphyloccus epidermidzs, biotype 111. The other culture corresponded to a S. epidermidis biotype I.

The results of the indirect immunofluorescein reaction and of the indirect immunoperoxidase- antiperoxidase reaction done on the S. epidermidis isolated from the cancer patients are illustrated by Figs. 4 and 5 respectively. The doughnut- shaped reaction of the CG-like antigen on the membranes and/or capsule is clearly demonstrated in these microphotographs. Figures 6 and 7 show the corresponding negative “reagent controls,”

of the two

FIG. 1. “Positive cell control.” BeWo cell, nonclonal human malignant trophoblast. Indirect immunofluorescein reaction with antiserum to the &subunit of CG as first antibody. Note the intensity of the immunohistochemical reaction at the level of the plasma membrane ( X 1520).

~i~~~~~ 8 and 9 illustrate the

demonstrate the specificity of the entire technique, in these examples, the indirect immunofluorescein reaction. The high concentration of the antigen, choriogonadotropin, at the level of the plasma membrane in the human malignant trophoblast is notable (Fig. 1).

In contrast, the nonmalignant human prostatic cells showed an absolute lack of reactivity (Fig. 2). Since the indirect immunofluorescein reaction is capable of detecting a minimum of one to two thousand molecules of antigen in the p l a ~ m a l e m m a , ~ ~ the lack of reactivity of this “negative cell control” demonstrate that the nonmalignant prostatic cells have only a minute expression of the CG-like antigen and/or do not express the information for its biosynthesis at all.

Figure 3 illustrates the results of one of the standard controls for the specificity of the second antibody, in this experiment, the fluorescein-tagged antibody. A complete negative reaction was obtained because the first antibody (specific antirabbit antiserum to the trophoblastic hormone) was replaced by PBS, and the second antibody is a fluorescein-labeled goat antirabbit antiserum. Since the cell used in this experiment Was the CG-producer malignant trophoblast, the results demonstrate that, in spite

F,c, 2, cell control,^^ MA-169, human nonmalignant prostatic cells. Indirect immunofluorescein reaction with antiserum to total CG as first antibody ( X 1520).


Tables 1 and 2 were also completely negative for both immunohistochemical reactions, thus demonstrating the lack of and/or extremely low expression of the information for the synthesis of the CG-like antigen.

Possible morphologic differences between the CG-like protein producing “cancer associated bacteria” and the “normal” non-producing forms were analyzed by a serial study of their ultrastructural characteristics by transmission electron microscopy (Figs. 14-21). Only an in- creasing thickness of the cell wall of the CG-like protein producers was apparent.

Gas chromatographic analysis of short and long chain fatty acids, used as “finger~rinting”’~ did not reveal any difference between the “producers” and the “nonproducers. ” Acid fastness was not demonstrated by the CG-like protein producing bacteria.

DISCUSSION

By the use of a specific antibody to 0-CG as well as to the complete hormone, CG, and the application of the highly sensitive indirect fluorescein-labeled and peroxidase-labeled antibody FIG. 3. “Reagent control” for specificity of the second

antibody, BeU’o cell. Indirect immunofluorescein reaction. First antibody was replaced by PHS. Negative results demonstrate that in spite of great production of the antisen, nonspecific attachment of the Huorescein-tagged second antibody to the cell does not occur ( X 1520).

techniques (sandwich technique, Or anti-

immunohistochemical reactions performed with ATCC 13637 strain of P. maltophilia and figs. 10 and 1 1 their corresponding reagent controls. The presence of the CG-like antigen is clearly evident, thus demonstrating the synthesis of the trophoblastic-like protein (Figs. 8 and 9).

Two of the three microorganisms isolated by Dr. Affronti and his associates from patients with carcinoma of the colon were classified as Escherichia coli. These two strains also revealed an active synthesis of the CG-like protein (Figs. 12- 13). Their corresponding reagent controls were totally negative.

It is necessary to note at this point, that all the CG-like protein producing microorganisms classified as S. epidermidis showed Gram positive characteristics. In contrast, the P. maltophilia and E. coli were demonstrated to be Gram negative bacteria.

The third bacteria isolated from a patient with carcinoma of the colon were classified as heudomonas aeruginasa. This microorganism, as well as ATCC 12818 of Streptococcusfaecalis did not synthesize the trophoblastic-like protein, since both immunohistochemical reactions were totally negative. T h e “control bacteria” listed in

FIG. 4. S. epzdermzdzs, biotype 111, isolated from urine of patient with metastatic carcinoma. Indirect immunofluorescein reaction with antiserum to the P-subunit of CG as first antibody, N~~~ the “doughnut” shape as well as the intensity of the reaction ( x 1520).

No. 4 CG-LIKE PROTEIN I N BACTERIA Aceuedo et al . 1223

body technique), we have demonstrated the presence of a membrane (capsule)-associated CG-similar or -identical immunoreactive protein in twelve samples of Staphylococcus epidermidis, two samples of Escherzchia coli, and ATCC 13637 strain of P. maltophzlia.

In addition to the synthesis of the CG-like common antigen, the only other common fea- ture of such a diverse group of bacteria uas the fact that every one of these microorganisms was isolated ,from patients bearing malignant neoplasms.

O n the other hand, the absence of the CG-like antigen in the Pseudumunas aeruginosa isolated from a patient with cancer of the colon as well as in ATCC 12818 strain of S. faecalis, demonstrated that nut eriery “cancer associated bacteria” has the capability to synthesize the trophoblastic- like protein at least in detectable amounts. T h e negative results also obtained with a great number of “noncancer control” bacteria, strongly supported the idea that the existence of these CG-like protein producing microorganisms is not a ubiquitous finding.

The demonstration of the de nuuo bacterial biosynthesis of a glycoprotein so similar to the human trophoblastic hormone that it shares not only antigenic sites but also membrane receptor t,inding si tes , as in the of ATCC 13637 p, maltophilia, 39 has tremendous biological implications. It may become one of the most important

FIG i. s epdermtdu. biotype I . isolated from tumor of patient with metastatic adenocarcinoma of breast. Indirect immunoperoxidase-antiperoxidase reaction with antiserum lo hCX as first antibody. Note the intensity of the immunohistochemical reaction at the level of plasma membranes (cell wail anti/or capsule) ( X 1600).

FIG. 6-7. Reagent controls of Figs. 5 and 6 for specificity of the second antibody. First antibody was replaced by PBS. The negative results indicate the absence of nonspecific attachment of the second antibody to the cells in the absence of the specific first antibody ( X 1520 and X 1600).


discoveries in the biomedical field if the biosynthesis of significant amounts of the glyco-protein is proven to occur only in bacterial strains that are growing in or have been exposed to the presence of cells and/or tissues associated with ectopic production of the CG-like protein, in other words, with cancer cells.

The explanation of the phenomenon is quite difficult, Since until now the biosynthesis of the protein(s) has been proved only in strains growing in the presence of CG-producing cells, the potential of “genetic exchange” with subsequent expression of the mammalian gene by the bacterial cells becomes a n interesting possibility.

Nevertheless, such natural genetic recombina- tion is difficult to explain for several reasons. One of them, for instance, is that CG is a sia- loglycoprotein with a molecular weight of about 40,000, constituted by two asymmetrical sub- 4 =

Fie: 8. I’. niultuphzlia, ATCC 13637 strain, isolated from the oropharyngeal region of a patient with mouth cancer. Indirect immunofluorescein reaction with antiserum to the &subunit of hCG as first antibody. The intensity of the immunohistochemical reaction is notable at the peripheral level (X 1520).

FIG. 9. P. maltuphdia, ATCC 13637 strain. Indirect peroxidase- anti-peroxidase reaction. Again note the intensity of the reaction at the level of the bacterial wall. ( X 1600).

No. 4 CG-LIKE PROTEIN IN BACTERIA Aceuedo et a/ . 1225

FIGS. 10-1 1. Reagent controls of Figs. 8 and 9 for specificity of the second antibody 1;irst antibody was replaced by PBS ( X 1520 and X 1600).

FIG. 12. E . colz, isolated from tumor of patient with carcinoma of colon. Indirect immunofluorescein reaction with antiserum to total CG. Note the intensity of the reaction in Frc;. 13. I<. rolz, as above. Indirect peroxidase-anti- the cell wall ( X 3040). peroxidase reaction ( X 1600).


FIGS. 14-17. Electron microscopy ultrastructural characteristics of a mitotic. cycle of “CGlike protein producers” S. p p i h n i d z , , biotype 111, (Figs. 14, 16, 18, 20) as compared to that of the “non-producers” (Figs. 15. 17, 19, 21) . The preparations were ohtained from a 15-hour culture. It can be noted that theonly apparent difference is the thickness of the cell wall (original magnification. X t00,OOO). Fiq. 14-upper left; Fig. 1.5-upper right; Fig. 16-lower left; Fig. 17-lower right.

units not covalently linked. Moreover, the available information concerning the biosynthesis of the hormone by the human trophoblast as well as by cancer cells in tissue culture’6~26~45~48 sug-

gests that the two subunits are made by separate genes. A natural process is apparently needed, which would involve plural recombinant events.

Adelberg, in his Report to the Recombinant

No. 4 CG-LIKE PROTEIN IN BACTERIA Aceuedo et al.

F~c;s. 18-21. See legend for Figs. 14-17. Fig. 18-upper left; Fig. 19-upper right; Fig. 20-lower left; Fig. 21-lowrr right.

1227

1228 CANCER

DNA Molecule Program Advisory Committee of the NIH4 also suggested that the phenomenon may also be explained if the gene coding for the CG-like protein is normally present in all bacteria as a result of conservation during evolution or as as example of convergent evolution.

Such an explanation may be supported by the demonstration that bacterial and mammalian cell membranes possess a number of glycopro- teins which are immunochemically and biologically cross-reactive. 31 For instance, ABH (0) substances, known to be present in human epi- thelial secretions, are also present in over half of some of 300 species of Gram negative organisms in the same or closely related chemical forms.

The explanation is also supported by our own work demonstrating the synthesis of the CG-like protein by cancer cells in rats and These rodents do not synthesize CG during their gestation. The synthesis of the trophoblastic-like protein that takes place after the malignant transformation occurred left no doubt that the coding for the glycoprotein synthesis was available and that it was very possible that malignant transformation was the event that derepressed or activated such information.

Such explanation in the case of the bacteria will require the existence of a repressing com- ponent or absence of a positive effector molecule in an organism that, as far as we know, only contains one double strand of DNA. One will have to consider therefore, mRNA directives as an intrinsic part of such a mechanism, if it does exist.

The results that we have obtained leaves little doubt of the existence of a biological association among the bacteria, cancer cells and spermatozoa through the presence of a common mem-

April 1978 Vol. 41

brane antigen. The cancer cells and spermatozoa are the only other cells in which such membrane-associated protein has been local- ized. This protein has been demonstrated to be immunologically and biologically similar to choriogonadotropin, CG, the glycoprotein hormone synthesized by mammalian tropho-

The biological and physiological implications of such findings in respect to cancer etiology, diagnosis, prevention and treatment are obvi- ous. The impact of these findings in the fields of oncology, bacteriology, epidemiology, genetics and molecular biology is so great that a detailed description will be beyond the scope of this com- munication. Since none of the current theories relating to these fields can explain the phenomenon observed with the bacterial systems, it is apparent that this phenomenon exposes the need for a new approach to the analysis as well as to our current concepts of cancer.

Finally, the feasibility of a practical application may be one of the most significant aspects of our investigations. The presence of a common membrane antigen expressed or detectable only in cancer cells, spermatozoa, trophoblasts and these “cancer associated” bacterial strains, exposes the potential for a specific immunological approach to the prevention and treatment of the disease as well as for fertility control. A bacterial vaccine elaborated with these CG-like protein producing microbes may theoretically produce antibodies sharing antigenic determinants against the common membrane antigen. The significance of this possibility in the fields of cancer control and reproduction control is obvi-

blasts, 1-3,9,35,42

ous.

REFERENCES

1. Acevedo, H. F., Slifkin, M., Pouchet, G. R., and Rak- hshan, M . : Human chorionic gonadotropin (hCG) in cancer cells. I. Identification in zn uztro and in vivo cancer cell systems. Proc. 3rd. Int. Symp. DPCa., New York, April 26-29, 1976. Abst. 356.

2. Acevedo, H . F., Slifkin, M., Pouchet, G. R., and Rak- hshan, M. : Choriogonadotropin (CG) in cancer cells. 111. Further studies in zn mtro and in vzuo cell systems. Prog. 58th Annual Meet. The Endocrine Society, San Francisco, CA, June 23-25, 1976. Abst. 420.

3. Acevedo, H . F., Slifkin, M., Pouchet, G. R., and Rak- hshan, M. : Identification of the @-subunit of choriogonadotropin (CG) in human spermatozoa. In The Testis in Nor- mal and Infertile Men. P. Troen and H . R. Nankin, Eds. New York, The Raven Press, 1977, pp. 185-192.

4. Adelberg, E. A. : The production of a human hormone (chorionic gonadotropin) by bacteria isolated from cancer patients: A possible instance of gene transfer in vivo between human and bacterial cells. In Report to members of the Recombinant DNA Molecule Program Advisory Committee of the NIH. Bethesda, January 6, 1977.

5. Affronti, L. F., Grow, L., and Begell, F.: Character- ization of bacterial tumor isolates. Fed. Proc. 34(3): 1043, 1975. (Abst. 4676.)

6. Affronti, L. F., Grow, L., Brumbaugh, R., and Orton, K . : Bacterial tumor isolates. Abst Ann. Meeting Am. SOC. hlicrobiol., Atlantic City, NJ, May 3-8, 1976. Abst. E-56.

7. Affronti, I,. F., Grow, L., Brumbaugh, R. , and Orton, K. : Production of human gonadotropin-like substance by bacterial tumor isolates. Abst. Ann. Meeting Am. SOC. Mi- crobiol., New Orleans, hlay 8-13, 1977. Abst. E-23.

8. American Type Culture Collection Catalog of Strains I , 12th Edition. H. D. Katt, Ed. Rockville, hID.. 1976.

9. Asch, R. H., Fernandez, E. O., and Pauerstein, C. J . : Immunodetection of hCG-like substance in human sperm. Frrlzl. Slerzl. 28:1258-1262, 1977.

10. Bain, J., and Ezrin, C . : Immunofluorescent localization of the LH of the human adenophypophysis. 3. Clin. Endocrul. Melab. 30:181-184, 1970.

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immunohistochemical localization of a choriogonadotropin ... 1978... · immunohistochemical...

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