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The Search for the Human Mammary Tumor Virus

By Elisabeth Rieping / Last update by Elisabeth Rieping 2004/12/12

Since it became known that the breast cancer of the mouse is transmitted by a virus, scientists had tried to discover a similar agent
responsible for the disease in women.

Of cause, the standard procedure to find a transmissible agent, a transfection experiment to an other person for ethical reasons was
not done.

But it was tried to transmit the human breast cancer to mice. In the animals infected with human breast cancer material the
incidence in mammary tumors raised only slightly from less then 1% to 3%. Instead an increased incidence of lymphoma could
become demonstrated after inoculation with human mammary cancer extracts[i]. More recently, the frequent development of murine
T-cell lymphomas after implantation of human inflammatory breast cancer cells in nude mice was demonstrated[ii]. Two
experiments that hint to a transmissible agent inside breast cancer material.

It has also been looked for immunological cross reactions with the mouse mammary tumour virus MMTV antigens in material of
human breast carcinoma lines [iii], pleural effusion fluids from patients [iv], in cancer tissue[v], and in human milk [vi]. And it was
looked for nucleic acids similar to those of MMTV [vii], [viii]. But early experiments might have been confounded by endogenous
viruses.

Then, monocytes from breast cancer patients were discovered to form giant cells [ix]. As giant cell formation may be a sign of
viral infection, it was tried to find retroviral particles and to test them for reverse transcriptase activitity.

After cultivating mononuclear cells for some days particles could be harvested from 97% of all breast cancer patients and from
about 11% of healthy controls. Possibly women, who were carriers, but who did not yet had developed cancer.
 The reverse transcriptases of the retroviruses work best with magnesium ions while cellular enzymes prefer manganese ions. The
enzyme of the particles from cultivated monocytes of breast cancer cells preferred magnesium ions. The particles were described to
be slightly smaller than MMTV. Its size was described to be more similar to that of Human Immundeficiency Virus HIV [x].

Two groups tried to confirm the work, but failed in different ways. One could not cultivate the monocytes in a way to produce
multinuclear giant cells. Neither did they find reverse transcriptase activity[xi]. The test done with the same primer that had been
used in the original work showed no reverse transcrpitase activity with the examined material from cultivated monocytes of human
breast cancer cells. It neither showed significant activity with HIV-1 nor with HTLV1 used as positiv controls. Why not was not
elucidated.

Contrary to this results, the other group found a lot of multinuclear giant cell formation. But they found them not only in cultured
monocytes from breast cancer patients but as well in those of control subjects with no or with benign breast disease[xii].

Fortunately this publication was very detailed. And one change in the design of their study in comparison to the original work was
the use of a different tissue culture medium. They had taken pooled female AB serum. Al-Sumidaie and his colleges had used
calves serum. Unpooled serum derived from one animal is much less likely to be contaminated.

In the original study it was shown that the retrovirus like particles could be cultivated from cells of nearly all breast cancer
patients and from the blood of about 10% of controls, possibly women who were already infected, but who did not yet have a tumor
big enough to be clinically detectable.

Pooled human serum might contain material from this virus carriers.

Very interesting was an additional result from this study. In spite of giant cell formation a reverse transcriptase activity could not
be demonstrated. The crude supernatant from the mononuclear cell cultures even blocked the activity of MMTV reverse
transcriptase which had been used as a positive control.
 As the originally described particles could be observed only after several days of cultivation in non human serum conditioned
tissue culture medium, it is possible, that by growing the cells with the help of pooled human serum the virus production or release
to the medium was inhibited or their activity like that of the MMTV particles became blocked.

It is known that some retroviruses seem to escape eradiction by producing little virus in vivo[xiii].

In her recent review on a possible retroviral etiology of human breast cancer [xiv] the author, probably stimulated by her
experience with bone disease [xv], [xvi], but also by examples of other animal [xvii],[xviii] and human retroviruses[xix] pointed out
that blood mononuclear cells should be examined first, when searching for a human breast cancer virus and that a possibly
causative retrovirus must not be MMTV or a very close relative.

Unlike MMTV which ist difficult to be seen as an agent getting entrance to the human food chain or another way of
transmittance, bovine leukemia virus BLV is a virus contained in milk used for human nutrition especially for nutrition of human
and pet animal infants in Western, but today also in non -Western societies. It had been suspicious since it was known. But with old
methods antibodies against it could not be found in human sera.

This has changed however[xx]. Using immunoblotting to test the sera of 257 humans for antibodies of four isotypes (IgG1, IgM,
IgA, and IgG4) to the BLV capsid antigen (p24), at least one antibody isotype reactive with BLV was detected in 74% of the human
sera tested. The specificity of the reactivity was strongly suggested by competition studies and by ruling out cross-reacting
antibodies to other chronic human viruses.

I was also looked for changes BLV might induce in bovine mammary tissue. Changed growth properties of bovine mammary
epithelial cells lines containing BLV DNA were found. The cell line showed reduced population-doubling time, higher saturation
density, and increased longevity, features that are typical for tumor cells[xxi]. That BLV could produce such changes in bovine
mammary cells inspired a search for BLV DNA in human breast cancer.

DNA of BLV was found in tissue of breast cancer patients. The study is going on[xxii].
 A search for traces of BLV but also for other bovine born agents in cultivated mononuclear cells of breast cancer patients might
thus be fruitful. Two known candidates propagating in cattle are the Bovine Immunedeficiency Virus BIV which is said to be quite
similar to Human Immune deficiency Virus HIV-1 and the Bovine Respiratory Syncytical Virus BRSV. And there may be other
bovine retroviruses which if not causing disease in their natural host may have gone unrecognised until today.

On the other hand, the argument that MMTV ist is not a likely candidate for entering the human food chain does not rule out its
involvement. Although it is not very likely that the virus jumped from mouse to man, it is possible that both species became
infected by the same food.

It was shown that the highest incidence of human breast cancer world wide occurs in countries where mus domesticus is the
resident native or introduced species of house mouse[xxiii].

Mus domesticus, also and better known as house mouse, is very interested in human food and it is not unlikly that it got infected
by human food or waste. That mice like man are probably new victims of a putative breast cancer inducing virus is likely because
they develop mammary tumors so easily.

In species harbouring a virus since ancient times it is unlikely that it still induces harmful disease. So while HIV – 1 could cause
millions of deaths in its new human host, its ancestors simian immundeficiency viruses found in numerous African species could
not become linked to diease symptoms. That mice develop mammary tumors so easily makes it likely that they contacted the
MMTV ancestors not to long ago.

Similar interesting is the observation of a 44% reduction in the incidence of breast cancer in women fully immunosuppressed
following organ transplantation .This was discussed in connection with observations that some kinds of immunsupression reduced
the incidence of mammary tumors in mice infected with MMTV, too[xxiv].

As mentioned above, retroviral DNA similar to MMTV DNA had been described in material from breast cancer patients before.
But is was difficult to distinguish it from endogenous sequences similar to retroviral DNA occuring in the human genome.
 Recently DNA sequences related to the env region of the MMTV genome became isolated from human breast cancer[xxv]. They
were specific for cancer cells and not found in genetic material from probably healthy breast tissue obtained from reduction
mammoplasty [xxvi]. They were neither present in healthy breast tissue of the same patient. That speaks for an exogenous origin of
the material.

MMTV like LTR and LTR env gene sequences were found too in human breast cancer cells but not in healthy tissue[xxvii]. They
contained the DNA for the glucocorticoid resposive elements GRE and the superantigen SAG.

That MMTV contains sequences coding for a superantigen is well known and the role the superantigen plays in the disease
developement is very well documented. So finding sequences conding for a superantigen reminds of MMTV.

But the Human Immundeficiency Virus HIV-1, too, containes sequences coding for an superantigen[xxviii].

Another group could, too, could demonstrate MMTV-like env gene sequences in human breast cancer DNA and in a T-Cell
lymphoma of a breast cancer patient[xxix].

The last result is interesting because in many inbred strains of mice, both breast cancer and T-cell lymphoma are caused by
MMTV [xxx],[xxxi],[xxxii].

To induce a lymphoma instead of a mammary tumor in the mouse changes in the long terminal repeats LTRs seem to have had
occurred constituing of about 400 nucleotide deletions and occasional substitutions resulting in unique tandem repeats. Viruses with
this changes in its LTRs cause lymphomas but no mammary tumors 4 to 11 months after inoculation[xxxiii].

As mentioned above, tumors induced by breast cancer material in mice were mainlyy lymphomas, too.

Very recently it was shown that progression from normal breast pathology to breast cancer is associated with increased
prevalence of MMTV like sequences in women and in men[xxxiv].
 A new argument against the involvement of MMTV in human breast cancer is the lack of a suitable receptor for MMTV in human
cells [xxxv]. The human transferrin receptor 1, whoose mouse homolog is the entry receptor for MMTV, does not work with
MMTV [xxxvi].

But it is interesting that individuals being homozygous for certain mutations of the transferrin receptor 1 gene in combination with a
variant of the hemochromatosis gene have an increased susceptibility to breast cancer[xxxvii].

The transferrin receptor 1 delivers iron-bound transferrin to cells. It is a membran-spanning glycoprotein with a short cytoplasmic
domain. At neutral ph it has a high affinity for iron loaded transferrin. After binding transferrin it traffics to the early acidic
endosome where it releases the iron. Then it goes back to the cell surface, where it releases the transferrin. Peptides or particles that
bind at other sites of the tranferrin receptor 1 cause it to be endocytosed and trafficed to the acidic endosome, too.

That mutations in the transferrin receptor change the susceptibility to breast cancer can thus be seen as an argument for the
possibility of a viral etiology of the disease, which does not have to be MMTV, but may be a relative better adapted to the human
homolog of the trasferrin receptor1 which seem to differs for less than 15 base pairs from it’s mouse counterpart [xxxvi] .

Going back to the epidemiology of human breast cancer there might be another possibility for a foreign virus to change it host
range . As milk products used for infant nutrition are cooked, it is unlikely that the infection takes place by virus particles
containing not very stable RNA, but as a transfection by heat stable proviral DNA contained for example in the DNA of milk
lymphocytes seems possible. By passaging through this DNA infected cells host range variants might develope which are able to
infect other human cells.

Experimentally host range variants of MMTVs were developed by serial virus passage in feline, human, rat, and canine cells. This
MMTV variants retained the ability to replicate well in the cells of its new species[xxxviii].
 On the protein level, material cross reacting with retroviral envelope antigens, which was not present in normal cells, was found,
too. This antigens cross reacted with antibodies against human immundeficiency virus type 1 HIV-1[xxxix]. But they were not
identical to HIV-1 proteins. Infection with HIV-1 was excluded by lack of reactivity to HIV-1 p24 another protein of HIV-1.

HIV-1 is related to human t-cell lymphothrophic virus HTLV-1 and its close relative BLV, the up to now best candidate for a
Human Mammary Tumor Virus HMTV. But HIV-1 seems to be even more closly related to the above mentioned BIV.

MMTV, HIV-1 are well examined viruses. Both are transmitted by milk and have structures coding for superantigens. HTLV-1
causing human disease also gets examined.

From the closely related bovine viruses only BLV is better known. BIV, BRSV and other bovine viruses are not. That breast
cancer contains material that can elicit lymphoma like MMTV, HTLV-1 and BLV and that it may contain exogenous DNA related
to MMTV and its superantigen coding sequences and exogenous proteins that cross react with HIV-1 hints to a may be still
unkown, closely related virus which considering the epidemiology might be of bovine origin.

That the DNA of BLV is found in the DNA of breast cancer tissue, makes this virus the most likely candidate. Especially because
the appearance of this virus in breast cancer tissue may explain the peculiar epidemiology of human and animal breast cancer, too,
as already proposed in 1986 [xl].

Since an increase in mammary tumors in breast fed daughters from breast cancer patients has never been observed [xli],[xlii] ,
[xliii], the putative virus is not very likely to produce infective particles in human epithelial breast tissue which can become
transmitted by milk.

So it may be easier to isolate it from cultivated monocytes of breast cancer patients, as has been done before [x].
[i] Basombrio M. A., Mayer A. M. S., Rivell C. An increased incidence of lymphoma in mice inoculated with human breast cancer
extracts. Arch. Geschwulstforsch., 47: 679-684, 1977

[ii] Wakasugi H., Koyama K., Gyotoku M., Yoshimoto M., Hirohashi S., Sugimura T., Terada M. Frequent development of murine
T-cell lymphomas with TcR/+, cd4/8- phenotype after implantation of human inflammatory breast cancer cells in BALB/c nude
mice. Jpn. J. Cancer Res., 86: 1086-1096, 1995.

[iii] Keydar I, Ohno T, Nayak R, Sweet R, Simoni F, Weiss F, Karby S, Mesa-Tejada R, Spiegelman S. Properties of retrovirus-like
particles produced by a human breast carcinoma cell line: immunological relationship with mouse mammary tumor virus proteins.
Proc Natl Acad Sci U S A. 1984 Jul;81(13):4188-92

[iv] Soule HR, Linder E, Edgington TS. Membrane 126-kilodalton phosphoglycoprotein associated with human carcinomas
identified by a hybridoma antibody to mammary carcinoma cells.Proc Natl Acad Sci U S A. 1983 Mar;80(5):1332-6..

[v] Chopra HC, Feller WF. Viruslike particles in human breast cancer. Tex Rep Biol Med. 1969 Winter;27(4):945-53

[vi] Moore DH, Charney J, Kramarsky B, Lasfargues EY, Sarkar NH, Brennan MJ, Burrows JH, Sirsat SM, Paymaster JC, Vaidya
AB. Search for a human breast cancer virus. Nature. 1971 Feb 26;229(5287):611-4.

[vii] Szakacs JG, Moscinski LC. Sequence homology of deoxyribonucleic acid to mouse mammary tumor virus genome in human
breast tumors. Ann Clin Lab Sci. 1991 Nov-Dec;21(6):402-12.

[viii] Andersson ML, Medstrand P, Yin H, Blomberg J. Differential expression of human endogenous retroviral sequences similar
to mouse mammary tumor virus in normal peripheral blood mononuclear cells. Aids Res Human Retroviruses 1996 12: 833-40.

[ix] Al-Sumidaie AM, Leinster SJ, Jenkins SA Transformation of blood monocytes to giant cells in vitro from patients with breast
cancer. Br J Surg. 1986 Oct;73(10):839-42.
[x] Al-Sumidaie AM, Leinster SJ, Hart CA, Green CD, McCarthy K. Particles with properties of retroviruses in monocytes from
patients with breast cancer. Lancet. 1988 Jan 2-9;1(8575-6):5-9.

[xi] Hallam N, McAlpine L, Puszczynska E, Bayliss G. Absence of reverse transcriptase activity in monocyte cultures from patients
with breast cancer. Lancet. 1990 Oct 27;336(8722):1079.

[xii] Kahl LP, Carroll AR, Rhodes P, Wood J, Read NG. En evaluation of the putative human mammary tumorvirus associated with
peripheral blood monocytes. Br J Cancer 1991, 63:534-540.

[xiii] Merezak C, Pierreux C, Adam E, Lemaigre F, Rousseau GG, Calomme C, Van Lint C, Christophe D, Kerkhofs P, Burny A,
Kettmann R, Willems L. Suboptimal enhancer sequences are required for efficient bovine leukemia virus propagation in vivo:
implications for viral latency. J Virol. 2001 Aug;75(15):6977-88.

[xiv] Labat ML Possible retroviral etiology of human breast cancer. Biomed Pharmacother. 1998;52(1):6-12.

[xv] Labat ML A new approach to the study of the origin of genetic diseases: retroviral etiology of osteopetrosis. Biomed
Pharmacother. 1991;45(1):23-7.

[xvi] Labat ML. Retroviruses and bone diseases. Clin Orthop. 1996 May;(326):287-309.

[xvii] Burny A, Bruck C, Chantrenne H, Cleuter Y, Dekegel D, Ghysdael J, et al. Bovine Leukemia Virus : Molecular Biology and
epidemiology. In Klein G. ed. Viral Oncology New York : Raven Press 1980: 231-89

[xviii] Miller JM, Miller MD, Olson C, Gilette KG, Virus-like particles in phythemagglutin.stimulated lymphocyte cultures with
references to bovine Lymphosarcoma. J Natl Cancer Inst 1969, 43: 1459-62.

[xix] Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles
from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A. 1980
Dec;77(12):7415-9.
[xx] Buehring GC, Philpott SM, Choi KY A reverse transcriptase assay for detection of the bovine leukemia virus. Am J Vet Res.
1977 Nov;38(11):1739-44. Humans have antibodies reactive with Bovine leukemia virus. AIDS Res Hum Retroviruses. 2003
Dec;19(12):1105-13

[xxi] Motton DD, Buehring GC. Bovine leukemia virus alters growth properties and casein synthesis in mammary epithelial cells. J
Dairy Sci. 2003 Sep;86(9):2826-38.

[xxii] Buehring GC. Bovine Leukemia Virus Infection and Human Breast Cancer Risk.
http://www.cbcrp.org/research/PageGrant.asp?grant_id=1815 or http://www.erieping.de/Buehring.htm

[xxiii] Stewart TH, Sage RD, Stewart AF, Cameron DW. Breast cancer incidence highest in the range of one species of house
mouse, Mus domesticus. Br J Cancer. 2000 Jan;82(2):446-51

[xxiv] Stewart et al (1995) Lancet 346: 796-798

[xxv] Wang Y, Holland JF, Bleiweiss IJ, Melana S, Liu X, Pelisson I, Cantarella A, Stellrecht K, Mani S, Pogo BG. Detection of
mammary tumor virus env gene-like sequences in human breast cancer. Cancer Res. 1995 Nov 15;55(22):5173-9.

[xxvi] Wang Y, Melana SM, Baker B, Bleiweiss I, Fernandez-Cobo M, Mandeli JF, Holland JF, Pogo BG. High prevalence of
MMTV-like env gene sequences in gestational breast cancer. Med Oncol. 2003;20(3):233-6

[xxvii] Wang Y, Pelisson I, Melana SM, Holland JF, Pogo BG. Detection of MMTV-like LTR and LTR-env gene sequences in
human breast cancer. Int J Oncol. 2001 May;18(5):1041-4

[xxviii] Townsley-Fuchs J, Neshat MS, Margolin DH, Braun J, Goodglick L. HIV-1 gp120: a novel viral B cell superantigen. Int
Rev Immunol. 1997;14(4):325-38.

[xxix] Etkind P, Du J, Khan A, Pillitteri J, Wiernik PH. Mouse mammary tumor virus-like ENV gene sequences in human breast
tumors and in a lymphoma of a breast cancer patient. Clin Cancer Res. 2000 Apr;6(4):1273-8.
[xxx] Michalides R., Wagenaar E., Hilkins J., Hilgers J., Goner B., Hynes N. E. Acquisition of proviral DNA of mouse mammary
tumor virus in thymic leukemia cells from GR mice. J. Virol., 43: 819-829, 1982

[xxxi] Dekaban G. A., Ball J. K. Integration of type B retroviral DNA in virus-induced primary murine thymic lymphomas. J.
Virol., 52: 784-792, 1984

[xxxii] Dudley J., Arfsten A., Hsu C-H. L., Kosak C., Risser R. Molecular cloning and characterization of mouse mammary tumor
proviruses from T-cell lymphoma. J. Virol., 57: 385-388, 1986

[xxxiii] Yanagawa S, Kakimi K, Tanaka H, Murakami A, Nakagawa Y, Kubo Y, Yamada Y, Hiai H, Kuribayashi K, Masuda T, et
al. Mouse mammary tumor virus with rearranged long terminal repeats causes murine lymphomas. J Virol. 1993 Jan;67(1):112-8.

[xxxiv] Ford CE, Faedo M, Crouch R, Lawson JS, Rawlinson WD. Progression from normal breast pathology to breast cancer is
associated with increasing prevalence of mouse mammary tumor virus-like sequences in men and women. Cancer Res. 2004 Jul
15;64(14):4755-9.

[xxxv] Mant C, Cason J. A human murine mammary tumour virus-like agent is an unconvincing aetiological agent for human
breast cancer. Rev Med Virol. 2004 May-Jun;14(3):169-77.

[xxxvi] Ross SR, Schofield JJ, Farr CJ, Bucan M. Mouse transferrin receptor 1 is the cell entry receptor for mouse mammary tumor
virus. Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12386-90.

[xxxvii] Beckman LE, Van Landeghem GF, Sikstrom C, Wahlin A, Markevarn B, Hallmans G, Lenner P, Athlin L, Stenling R,
Beckman L. Interaction between haemochromatosis and transferrin receptor genes in different neoplastic disorders. Carcinogenesis.
1999 Jul;20(7):1231-3.

[xxxviii] Howard DK, Schlom J. Isolation of a series of novel variants of murine mammary tumor viruses with broadened host
ranges. Int J Cancer. 1980 May 15;25(5):647-54.
[xxxix] Rakowicz-Szulczynska EM, Jackson B, Szulczynska AM, Smith M. Human immunodeficiency virus type 1-like DNA
sequences and immunoreactive viral particles with unique association with breast cancer. Clin Diagn Lab Immunol. 1998
Sep;5(5):645-53.

[xl] Rieping E. Breast Cancer and Early Contact to Cow’s Milk. Cancer letters 30: June Supp. 104, 1986. Updated and more
explicitly under www.erieping.de

[xli]Morgan RW, Vakil DV, Chipman ML. Breast feeding, familiy history and breast disease. Am J Epidemiol 99: 117-122, 1974.

[xlii] Tokuhata GK. Morbidity and mortality among offspring of breast cancer mothers. Am J Epidemiol 89: 139-153, 1969.

[xliii] Titus-Ernstoff L, Egan KM, Newcomb PA, Baron JA, Stampfer M, Greenberg ER, Cole BF, Ding J, Willett W, Trichopoulos
D. Exposure to breast milk in infancy and adult breast cancer risk. J Natl Cancer Inst. 1998 Jun 17;90(12):921-4.

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The Search for the Human Breast Cancer Virus

Last update by Elisabeth Rieping 22.12.2004

Until today the search for a human breast cancer virus did not result in a reproducible method of isolation. Analysis of successful transmissions
show that there might be an agent that can become transmitted cell-bound causing lymphoma in mice. It is not unlikely that the agent is a provirus
harbored by mononuclear blood cells quite similar to HTLV-1 and BLV.

Ever since the discovery that breast neoplasms of the mouse are transmitted by a virus, scientists have tried to discover a similar agent responsible
for the disease in women.
The standard procedure to find a transmissible agent, a transfection experiment to another person, was not used, of course, for ethical reasons.

Instead researchers tried to transmit human breast cancer to mice. But in the animals infected with human breast cancer material, the incidence of
mammary tumors increased only slightly from less then 1% to 3%. However, a significantly increased incidence of lymphoma in the infected mice
was observed [i].

Aside from the abovementioned research, no other work concerning the transfection of human breast cancer to animals could be found. Presumably,
this is not because nobody tried it, but because nobody tried it successfully. So why was this group at least partially successful?

The usual procedure microbiologists use to distinguish between bacterial and viral agents is to filtrate the material. Of cause, this filtration also
blocks the transmission of cells.

But these successful researchers prepared their human breast cancer material differently. They homogenized it, added antibiotics to kill
contaminating bacteria, centrifuged it and extracted the interphase between cells and fat. Using this procedure, their material will have contained
enough cells to transmit some latent proviruses integrated in the DNA of these cells. If they had used the usual procedure of filtering, cell-bound
viruses would not have been transmitted.

It is likely that other researchers who were not successful in transmitting the breast cancer virus used filters as this was then standard procedure. At
that time it was not known that some retroviruses, for example the bovine leukemia virus BLV, seem to try to escape eradication by producing little
virus in vivo [ii]. Viruses like BLV or the human immune deficiency virus, HIV, which are transmitted by live cells, were not yet well known in
1977.

More recently, in 1995, the frequent development of murine T-cell lymphomas after implantation of human inflammatory breast cancer cells in
nude mice was demonstrated [iii].

These two experiments hint at the existence of a transmissible agent in breast cancer material. Probably both experiments worked because actual
cells were used for the transfection, although in the first experiment cells were probably used unintentionally.

Already in 1986, monocytes from breast cancer patients had been discovered to form giant cells [iv]. As giant cell formation may be a sign of viral
infection, attempts were made to find retroviral particles and to test them for reverse transcriptase activity.
After cultivating mononuclear cells for some days, particles could be harvested from 97% of all breast cancer patients and from about 11% of
healthy control group participants. It is possible that this 11% represents women who were carriers, but who had not yet developed cancer.

The reverse transcriptases of retroviruses work best with magnesium ions, while cellular enzymes prefer manganese ions. The enzyme of the
particles from cultivated monocytes of breast cancer cells preferred magnesium ions. The particles were described to be slightly smaller than the
murine mammary tumor virus MMTV. Its size was described to be more similar to that of HIV [v].

The work of this group from Liverpool was not continued because two other groups had unsuccessfully tried to confirm the work. They both failed
in different ways.

One group could neither cultivate the monocytes in a way to produce multinuclear giant cells, nor could they find reverse transcriptase activity[vi].
These test, done with the same primer that had been used in the original work, showed neither reverse transcrpitase activity with the material from
cultivated monocytes of human breast cancer cells, nor with HIV-1 or with HTLV-1 which were used as positive controls. No information was
given by the researchers to elucidated why this was so.

Contrary to these results, the other group found significant multinuclear giant cell formation. But they found them not only in cultured monocytes
from breast cancer patients, but also in those of control subjects with no or with benign breast disease [vii].

Fortunately this publication was very detailed. One change in the design of their study in comparison to the original work, they wanted to reproduce,
was the use of a different tissue culture medium : pooled female AB serum. In the successful experiments published in 1988 calves serum had been
used. Unpooled serum derived from one animal raised in a controled environment is much less likely to be contaminated.

As the original Liverpool study had shown that the retrovirus like particles could be cultivated from the blood of about 11% of healthy controls,
possibly women who were already infected, but who did not yet have a tumor big enough to be clinically detectable, pooled human serum might
contain material from theses virus carriers.

An additional result from this study is very interesting. In spite of giant cell formation, a reverse transcriptase activity could not be demonstrated.
The crude supernatant from the cell cultures even blocked the activity of MMTV reverse transcriptase, which had been used as a positive control.

As the originally described particles could be observed only after several days of cultivation in non-human, serum conditioned tissue culture
medium, it is possible that by growing the cells with the help of pooled human serum, the viruses produced by or released to the medium were
inhibited or that their reverse transcriptase activity - like that of the MMTV particles - was blocked.
In a recent review on a possible retroviral etiology of human breast cancer [viii] the author, probably stimulated by her experience with bone disease
[ix], [x] but also by examples of other animal [xi],[xii] and human retroviruses[xiii] pointed out that blood mononuclear cells should be examined
first when searching for a human breast cancer virus and that a possibly causative retrovirus must not necessarily be MMTV or a very close relative.

Unlike MMTV which is difficult to perceive as an agent accessing the human food chain (or another way of transmittance) bovine leukemia virus
BLV is a syncytia or giant cell forming virus contained in milk used for human nutrition, especially for nutrition of human and pet animal infants in
Western, but today also in non -Western societies. It has been suspected since its discovery, but with old methods, antibodies against it could not be
found in human sera.

This has changed however [xiv]. Using immunoblotting to test the sera of 257 humans for antibodies of four isotypes (IgG1, IgM, IgA, and IgG4) to
the BLV capsid antigen p24, at least one antibody isotype reactive with BLV was detected in 74% of the human sera tested. The specificity of the
reactivity was strongly suggested by competition studies and by ruling out cross-reacting antibodies to other chronic human viruses.

Independently and earlier than the above mentioned work, antibodies against BLV in humans had been demonstrated by an other group[xv] They
had looked for antibodies against BLV in patients with multiple sclerosis and found them both in patients and in controls. Therefore they
discontinued this work.

Changes which BLV might induce in bovine mammary tissue were also sought. Altered growth properties of bovine mammary epithelial cell lines
containing BLV DNA were found. The cell lines showed reduced population-doubling time, higher saturation density, and increased longevity,
features that are typical for tumor cells [xvi]. The evidence that BLV could produce such changes in bovine mammary cells inspired a search for
BLV DNA in human breast cancer.

DNA of BLV was found in tissue of breast cancer patients. The study is going on [xvii].

If BLV is the cause of human breast cancer and if the infection is transmitted by infant food contaminated with a bovine born virus, [xviii], it should
be traceable in about every tenth person in Western society since about one in ten women gets breast cancer.

In NewYork healthy blood donors are checked for human T-cell lymphothropic viruses HTLV-1 and HTLV-II, although the city is not an area in
which this viruses are endemic. About 8,6% of the blood donors diagnosed to be free of HTLV-I and II, as they lack antibodies against viral
structural proteins, were shown to have antibodies against a Tax protein homologous to the HTLV Tax Protein. DNA homologous to TAX DNA
was found by two independent groups as well [xix],[xx]. As New York is not an area in which HTLV is endemic, this was a surprising result.
It would not be surprising if this protein, homologous to HTLV-1 Tax, is the BLV Tax protein. BLV is a virus closely related to the HTLV-I and is
contained in the bovine milk used as food by humans.

BLV DNA is integrated in the DNA of bovine milk lymphocytes. It is likely that it is also in the pooled bovine milk used for the production of baby
food. As DNA is not destroyed by cooking, the integrated BLV DNA will not be destroyed by the heat based procedures used to make infant food
more safe. DNA is sufficient for transfecting the virus.

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Literature

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[xiii] Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and
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