THE INSTITUTE FOR CANCER RESEARCH
and

THE LANKENAU HOSPITAL RESEARCH INSTITUTE
7701 Burholme Avenue
Fox Chase
Philadelphia 11, Pa.
November 18, 195)

Professor Joshua Lederberg
Department of Genetics

The University of Wisconsin
Madison 6, Wiscensin

Dear Jashua:

We were delighted to hear from you that you will participate
in the Conference on Ascites Tumors in New York next May 19th and 20th.
Not only was there no trouble in justifying your appearance, but the meeting
weuld have to be considered very incomplete without discussion of our
general problems by a creative worker in the field of microbiological genetics.
As you say, some of the parallelisms which have been drawn between
mammalian tissue genetics and bacterial population problems have been rather
slopoy and far fetched. We are counting on you to dispel the fog.

It will probably ve ike somewhere in the program, to draw
out Lettré on the subject of mitochondrial recombination with granule-free
cells. We have seen considerable evidence in our daily handling of various
ascites tumors for incorporation of particulate matter by various types of
ascites cells, but have not done anything about it. Off-hand, I am not
adverse to accepting Lettré's story, which however, should be repeated more
critically. It belongs in the same chapter as "Incerporation of Chromatin
Fraction by Cells", Although, I am disinclined (because of my personal
experience with rather high-take percentages after inoculation of single
cells) toward believing with my friends, Paschkis and Cantarow, in trans—
duction by chromatin fraction, I do not feel that George Klein's genetic
evidence, published in CANCER RESEARCH in 1952, settles the issue entirely.

» published in 1952, or wet /f fe Resckkent

Your idea, suggested to George KleinYoF using a genetic
dependence or resistance marker in addition to histocompatibility, avpeals
to me very much, and some one should do this. The other day I leaned over
backwards to think of all the reasons for continuing with a little further
work on chromatin fraction, and I put these ideas down rather informally
for Dr. Paschkis,. A cepy of this discussion is enclosede You probably won't
like it.

Under separate cover two brief items of recent vintage are
being sent.for your reprint files,

With sincere good wishes and regards to Mrs. Lederberg,

Yours ever,

/eok

Theodore S. Hauschka
TSH/ ems

Enclosure
~ ‘9

~~ |

A.

Be

C.

De

iscussion of Klein's results after injection of
cnromatin-fraction of two mouse lymphomas into Fi hybrids

(See Cancer Research, 12:589-590, 1952)

Points in favor of Klein's argument that chromatin-fraction was probably
contaminated with 6C3HED or DBA lymphoma cells:

(1) Rapidity of appearance of tumors (10-27 days after inoculation of
ehronatinefraction). This tine element is comparable with Hauschka!ls

observations after i.p. inoculation of 20-30 6C3HED or DBA cells per
MOUSE.

(2) Transplantability of tumors "induced" in F, into parent type of tumor
origin.

Points weakening Klein's argument.

(1) The tunors which appeared in F, after the chromatin-fraction was injected
were (with a single exception) all solid tumors, while Hauschka obtained
100% ascites after inoculation of 20-30 intact viable cells.

(2) Exceptions are known to the genetic "rule" that F, tisme is transplantable

only into comparable F, hybrid hosts (See Little's review in "Genetics in
the 20th Century").

(3) Failure to detect intact cells or intact nuclei in chromatin-fraction.

Information not included in Klein's experimental scheme.

(1) Results after known small number of intact lymphoma cells is put into
F, mice. Are the resulting tumors solid or ascites? liow many cells are
needed to produce 100% takes in Fy mice within the time limits of the
chromatin«fraction result? Hausehka has found F, hosts more resistant
to low cell dosages than pure C3H or DBA mice.

(2) Results after tumor chromatin-fraction is put directly into susceptible
strain in which tumor originated. Are the resulting growths solid or
ascites?

Klein's experiments, while providing seemingly strong genetic evidence for the
contamination of tumor chromatin-fraction with a few viable cells are not,
therefore, entirely conclusive.

The sequence of events through which a normal Fy lymphocyte might be con-
verted into a malignant one, whieh then becomes transplantable into the parental
genotype providing the chromatin-fraction, involves:

(1) Ineorporation of functional chromatin elements into F, lymphocytes.
(Uptake of particulate material by mamnalian celis of several types, both
in vitro and in vivo is a well~esta>lished phenomenon. See, for instance,
lettre's recent results with Ehrlich ascites cells.)
(2)

(3)

(4)

(5)

=o

Change of at least one cell, but judging from the short latent period
several normal Fy lymphoc,;tes into neoplastic elements. This would
seem to presuppose that the maliynant change is inherent in a portion
of the chromosomal material of the chrouwatin=-fraction or in a contame
inating cytoplasmic entity. If functional chromosomal elements are
involved, it must be assumed further that - after getting into the

cell - these specific genetic entities are incorporated into the nucleus
and eyually distributed among daughter cells in subsequent mitoses,

Uptake of functional lymphosarcoma chromatin-fraction by normal F
lymphocytes, which have thereby become neoplastie, would also have

to neutralize the iso-antigenic character of those histoconpatibility
factors which entered into the susceptible F, mouse through the non-
susceptible parent strain, the latter being toos refractory to the

tunor furnishing the chromatin-fractione Granting the mechanical premise
of chromatin uptake, the possibility of antigenic modification by specific
isoewantigenic entities in the ehrouatin-fraction is compatible with our
demonstration (Hauschka, T.S., and Levan, A. Inverse relationship between
chromoso.e ploidy and host-specificity of sixteen transplantable tumors.
Exper. Cell Research, 4:h57-467, 1953) of a consistent inter-relationship
between aneuploidy and decreased transplantation specificity. incorpora-
tion of antigen-carrying chrosmatin-fraction could conceivably make an

Fy lymphocyte ansuploid with respect to the specific gene-action which
controls antigenic end-products and, thus, would tend to favor trans~
plantability beyond the linits of classical genetic expectation, at

least into the susceptible parental genotype.

The following cytologic data may be relevant -here (see Levan, 4., and
Hauschka, T. S. Nuclear fragmentation--a nomial feature of ine mitotic
cycle of lymphosarcoma cells. Hereditas, 391137#18, 1953): Although
nuclear fragnentation and lobation is a frequent phenomenon in sone

mouse lymphosarcomas, a mecha:ism exists for the reconstitution of a
Single normal metaphase plate, after the separate micronuclei have under=
gone synchronous prophases. Anaphase is normal and telophase culminates
again in nuclear lobation and fragmentation. sits of chrosatin material
with functional kinetochores, which manage to get into an F, lymphocyte,
might behave like the a»ove micronuclei and become part of the functional
genone of the cell, rendering it malignant and increasing its trans~
plantation range.

Jvbviously, a number of rather labored assucptions are needed to circunvent
Klein's classical genetic argument. It should be remembered, however,
that histocomupatibility genetics is no longer the watertight aggregate

of laws it was even three ,ears ago. Difficulties and the need for new
interpretation were introduced into the field by:

(a) the influence of heteroploidy on the antigenic specificity of
grafts (Hauschka);
~3-

(b) the F, adaptation phenomenon (Barrett et al., iiausechka)s;

(c) the in utero conditioning of mice to accept, in adult life,
persistent skin-grafts to which they are genetically refractory
(Billingham et al.);

(d) antige:ic conditioning of non-susceptible hosts by cell-free
enhancing substance (Kaliss, Snell et al.).

It, therefore, appears @qu@liy probable that Specific chromatinefraction
might so alter Fy cells, that they becone genetically conpstible with the
donor=type of the chromatin.

Suggestion for a possibly crucial experiment.

Insert tunor-chromatin-fraction into now type of Algire dise chanber and
sew into the peritoneal cavity of animal genetically conp.tible with the twsor
from wiich the fraction was prepared. Since dise does not (usually) perait
cells to pass, no tumor should arise inside the chanber if chronatin-fraction
contains no cells.

If the fraction is, on the other hand, containinated with viable cells,
then twnor should start to grow wit.in the chamber after a latent period.

Controls could ineludet

(1) Gmall :nown nunber of viable maliznant cells added to curomatin=fraction
inside disc.

(2) A few neoplastic cells only, inside disc.

(3) A mixture of normal lymphocytes plus malignant chronatinefraction inside
disc.

Theodore 3. Hauschka
Novenber ll, 1954