Journal of Cancer Therapy, 2011, 2, 181-189
doi:10.4236/jct.2011.22023 Published Online June 2011 (http://www.SciRP.org/journal/jct)
181
Normal Human Cell Conversion to 3-D
Cancer-Like Growth: Genome Damage,
Endopolyploidy, Senecence Escape, and Cell
Polarity Change/Loss
Kirsten H. Walen
California Department of Public Health, Viral and Rickettsial Disease Laboratory, Richmond, USA.
Received January 17th, 2011; revised April 11th, 2011; accepted April 18th, 2011.
ABSTRACT
In cell cultures monola yered cell growth is controlled by c ontact inhibition which again is controlled by the cell polar-
ity system by always being positioned in accord with the cytoskeleton axis. Presently, cycling endopolyploid cells
(tetraploidy) were shown to undergo perpendicular divisions relative to the cytoskeleton axis which disrupted to some
degree contact inhibition in the near-senescent phase of human primary cells. These experiments included genome
damage-induced endopolyploidization (TAS-treated) to simulate as a model system the state of in vivo accelerated cell
senescence (ACS) which is induced by therapy-associated genomic damage. From ACS delayed tumor re-growth (re-
lapse) occurs from “robust” cell propagation, but mechanisms for such cell escape from senescence are unknown. For
TAS-treated a karyoplast bud-off process with change to limited mitotic activity occurred in young senescent cultures.
In old, deep senescent (5 - 8 weeks) cultures, unexpectedly escape cell-growth showed three dimensional (3-D) tu-
mor-like spheres from growths of morphologica lly different cells as compared to th e fibrob lastic phenotyp e. These cells
expressed cell polarity change, and very condensed nuclei were variously perpendicularly oriented to what-ever cell
polarity was present. These results were discussed in regard to in vivo relapse and, to the importance of cell polarity
change in tumorigenesis. Induced senescence as an anti-tumor mechanism in therapy treatment becomes a questionab le
procedure from the present experimental results.
Keywords: Endo-Division Perpendicularity, Contact Inhibition, Deep-Senescence, Escape-Route, Amorphous Flat Cells
1. Introduction
Senescence of cells as an anti-tumor mechanism is con-
troversial for both cancer-therapy-induced accelerated
senescence (ACS) and telomere associated senescence
(TAS) [1-5]. Recent works have shown both in vivo and
in vitro reversions to proliferating cells [6-13]. ACS es-
cape cells, following a period of time-delay, were shown
to re-grow tumors with similar phenotypic tumor cells,
but showing increased growth rate and aggressiveness
[14-18]. How these sinister tumor cells gain propagating
robustness, and how cells escape from the large flat cells
which has a wrong nuclear/cytoplasmic ratio for mitotic
activity, are unknown. These problems for effective
therapy are difficult-to-impossible to assess for in vivo
studies. Therefore, in vitro model studies in search of a
cellular morphological base seem appropriate because, it
can be followed by genetic pathway studies in both in
vitro and in vivo. Such in vitro works for TAS has shown
that one way of escape from young senescent cells was a
karyoplast budding-o ff process [10-13]. These small cells,
wrapped in mother-cell membrane grew in cytoplasmic
content and entered mitosis, but with very limited pro-
pagating capacity (2 - 16 cell stage). Presently, it is un-
known if TAS escape cells express differential growth
capacity coming from young or old senescent cells. But
this possibility has some merit from observation of p53
activation and chromatin-associated DNA damage foci
following H2O2 treatment of deep (old) senescent cells
[19]. These experiments suggest that both time-delayed
ACS- and TAS-associated escape cells have undergone
repair of genomic damage during the protracted senes-
cence arrest-period. And thus, escape cells from old se-

Normal Human Cell Conversion to 3-D Cancer-Like Growth: Genome Damage, Endopolyploidy,
182 Senecence Escape, and Cell Polarity Change/Loss
nescent cells may show gain in growth capacity (see
Discussion).
This supposition brings in anoth er relatively new issue
in tumorigenesis which also influences growth capacity,
namely: change/loss in cell polarity contro l of growth [20,
21]. This change was found to be associated with
skewed/perpendicular cell division relative to the cy-
toskeleton axis, and is entering human tumorigenesis via
worms and Drosophila cancer studies [22,23 ]. Such divi-
sion led to cancer cell development, but the mechanism
behind perpendicularity is not known. It is however
molecularly accepted that cell polarity change occurs in
late tumorigenesis [22].
This peculiar positioning of the division plane occurs
for the unicellular radiolarian Aulachanta in vegetative
reproduction of endopolyploidy to offspring cells with
polarity change [24]. Perpendicularity is not known for
regular mitotic division, therefore, the idea of it being
tied to the endopolyploid irregular division system [25,
26] is being explored in the present study. In human tu-
mor progression there is a gradual increase in cells
showing morphological, architectural alterations, desc r ib ed
as mild, modest and severe dysplasia, which originate
from a dedifferentiation process of the original differen-
tiated (specialized) mother cell. The famous pathologist
von Hansemann, linked such cell alterations to “greater
capacity for independent existence” with possible change
to “complete spherical cells” [27]. In simple words such
cells have lost the original growth controlling cell polar-
ity system, and unscheduled proliferation (e.g., telom-
erase positive tumor cells) becomes a biological lawless
process.
The purpose/aim of the present study is to establish a
model system for senescence-associated escape cell-
events from young and old senescent cell cultures: are
the escape routes and the growth capacities the same?
But firstly, the presumed perpendicular endopolyploid
division in the presenescent phase must be verified and
assessed for associations to cell polarity loss/change.
Therefore, escape cell growths is not only monitored for
extended mitotic capacity, but also for cell morphologi-
cal changes of the strongly polarized fibroblastic pheno-
type.
In order to simulate with greater likeness of the TAS-
to the ACS-phase (from therapy-induced genomic dam-
age to kill cells), primary diploid cells in the pre-senes-
cent phase (3-6 population doublings from senescent flat
cell changes) were exposed to genomic damage in addi-
tion to that from broken telomeres (TAS-treated). In-
crease in cycling endopolyploid cells (from re-replication
of G2/G1 arrest escaped cells [28,29]) would precede
activation of the senescence-inducing p53/p21/p14 pro-
gram which are relatively slow processes in both in vitro
and in vivo systems [4,9]. The present data showed
karyoplast bud-offs and three dimensional (3-D) tu-
mor-like spheres with polarity changed cells, but only in
the old, deep senescent cell cultures. These results are
discussed regarding: DNA-repair synthesis in flat cells,
and how cell polarity change can be augmented by cells
going through a senescent phase.
2. Materials and Methods
The progressive culturing of diploid cell strains (L645
and WI-38 both from embryonic lungs) to the senescent
phase has been described earlier [13,30] which include
weekly passages at a 1 to 4 cell dilution factor. This
method results in ~50 - 60 population doubling which is
the Hayflick-limit [31]. The common Eagles’ basal me-
dium supplemented with 10% serum, penicillin/ strepto-
mycin and glutamine as cell growth medium was used.
Late passage cells, 3 - 5 passages (near-senescent phase)
before significant presence of non-proliferation capable
large senescent flat cells, were washed 2x in Hank’ bal-
anced salt solution. This was followed by a 1x-5-day
exposure to medium deficient in amino acid glutamine
(TAS-treated) [32,33]. Since 10% serum in such medium
revealed continued, but reduced presence of mitotic cells,
the amount was reduced to 2% for the present study.
Following the five day exposure, complete medium (10%)
was added for 1-2 days recovery period before tryp-
sin/versene transfer to new flask cultures. These cultures
were passaged with diminishing dilution until senescent
cells predominated the cell populations. Control cultures
(TAS-only) were passaged the same way, and consisted
of two cultures each of the respective primary cell strains.
The optimum survival for the treated cu ltures to reach 5 -
8 weeks in the senescent phase dependent on high cell
density and pH adjustments during maintenance media
changes. Low density cultures were therefore, combined
by careful trypsin/versene treatment since they did not
reveal sigificant re-growth from escape cells. Live cell
photography (film) at 100x from well-surviving cultures
was done by a VWR Vista Vision inverted, phase con-
trast microscope with a Pentax (2x-M) camera attached.
For endopolyploid perpendicular divisions pre-senescent
cells (both cell strains) were grown on chamber slides for
fixation at various harvest days which consisted of sev-
eral changes with 3 parts methyl alcohol to one part of
acidic acid (3:1 Carnoy’). The slides were air-dried,
stained with 2% Giemsa in phosphate buffer solution at
pH 6.8 and coverslipped with Coverbond. Photography
through a Zeiss Standard microscope with an attached
Zeiss camera for the following picture magnifications:
750x and 2475x for Figure 1 were done.

Normal Human Cell Conversion to 3-D Cancer-Like Growth: Genome Damage, Endopolyploidy,
Senecence Escape, and Cell Polarity Change/Loss
183
Figure 1. Perpendicular division relative to the cytoskeleton axis for mostly endo-tetraploid cells. (a)(b)(c): Interphase and
prophase nuclei in a perpendicular orientation. (c): This prophase cell contains an MTOC body situated on the upper-right
side of the nucleus (see text). (d)(e)(i): lateral as opposed to vertical separations of whole genomes. (f)(g)(h): perpendicular
orientation of telophase nuclei relative to surrounding normal fibroblastic growth. (e): upper division figure has a pear shape,
lower shows endopolyploid characteristic side-by-side telo-metaphases in the first meiotic-like reductional division, (i): sepa-
rations of genomes in the second division. (j): a sheet of cells mostly composed of (endo)-tetraploid cells with less striated cell
polarization compared to nomal fibroblasts which contain several perpendicular divisions. Scale bar = 30 μm (j), same mag-
nification (a)(c)(d) and (b)(e)(f)(g)(h).

Normal Human Cell Conversion to 3-D Cancer-Like Growth: Genome Damage, Endopolyploidy,
184 Senecence Escape, and Cell Polarity Change/Loss
3. Results
3.1. Pre-Senescence
Contact inhibition for normal fibroblast cells prevents
cells to grow into multilayered growth and maintains
nuclei/mitosis with apical/basal orientation in accord
with the cytoskeleton axis (vertical division). Figure 1
shows various key features/consequences of perpendicu-
lar endopolyploid divisions starting with interphase and
prophase nuclei being perpendicularly oriented to the
cell’ longitudinal axis (Figures 1 (a-c)). In Figure 1(c)
(prophase) there is a small round body located on the
upper-right side of the nucleus from which strands of
material is radiating. Presumably, this is a microtubular
organizing center (MTOC), but in the wrong po sition for
normal mitotic vertical division. Genomic separations
with so-called lateral moves for endopolyploid divisions
are clearly demonstrated in figures 1DEI. The cells be-
come pear-shaped (Figure 1(e)(i)) and in Figure 1(e) the
characteristic endopolyploid side-by side star-like telo-
metaphases are seen. The endopolyploid second division
[13,25,26] is also clearly demonstrated in figure 1I and
also perpendicularity of division products in relationship
to surrounding fibroblastic cell growth (Figures 1(e)(g)
(h)). This confirms the perpendicularity of single endo-
polyploid cell division. Lastly Figure 1(j) shows several
perpendicular divisions, in a cell sheet mostly composed
of (endo)-polyploid cells with less striated polarity as
compared to diploid cells. Taken together these few key
illustrations undeniably demonstrate perpendicular divi-
sions relative to the cytoskeleton axis of endopolyploid
genome separations which are derived from re-replica-
tion of genome damaged normal, diploid human cells.
The five-day exposure of pre-senescent cells to me-
dium deficient in glutamine caused a significant slim-
ming of the cells and mitotic cells became absent (Figure
2(b) compare Figure 2(a)). In recovery, complete me-
dium there was occurrence of synchronized mitotic ac-
tivity a few hours later (Figure 2(d)). Figures 2(c) and
(h) are growth foci with slight to moderate change in cell
polarity axis. Figure 2(h) shows high mitotic activ ity and
divisions to four products which are indicative of en-
doploid genome reduction divisions [25,26]. The change
to the senescent flat cell morphology was associated with
presence of large amounts of lysosomes (Figure 2(f))
which is a senescent cell marker [34] and was visibly
being extruded into the medium (see insert). The acidity
caused considerable cell death such that cultures had to
be combined for survival in deep senescencens (see M
and M). (These cultures did not show chromocenters and
lysosomes were reduced to a ring around the nuclei:
Figure 3(a)). Escaped karyoplasts became attached to
the cultural surface (Figure 2(g)) for mitotic activity, but
as earlier observed the propagation was very limited in
these young senescent cultures (not shown).
3.2. Escape Cell Growth from Deep
Senescence
In the old TAS-treated senescent cell cultures rarer
growths as multilayered spheres (3-D) were present.
These tumor-like balls were packed with cells (mostly
dead) and were both surface attached and free floating in
the medium (Figures 3(a-e)). Altogether thirteen varying
sized spheres were observed, but the rate of occurrence
was likely higher because, detached spheres were no
doubt lost through media changes. More recent observa-
tions for isolation purposes support this supposition. The
cell morphology had chan ged from the striated fibroblas-
tic phenotype to tri-quadri-shaped cells, and the nuclei
were variously oriented in these cells. The nuclei also
showed hyper-chromasia and were very condensed. A
beginning focus of such cells appears to be located on
top of an amorphous flat cell (Figure 3(a)). These cells
showed early-on the ability of growing on top of each
other (Figure 3(b)). Around the spheres there are these
cells and also cells with less polarity changed morphol-
ogy as compared to the fibroblast.
4. Discussion
The present bud-off karyoplast mechanism leading to
mitotic cells might be one answer to proliferation capable
cells before activation of telomerase in tumorigenesis
which is a late event [35]. The mechanism achieves es-
cape from anti-proliferation proteins in flat cell cyto-
plasm [36]. But it is not known to what degree such
karyoplasts can rebuild an effective growth controlling
cell polarity system. The limited growth from senescence
escape cells in young cultures and the apparent mixture
of polarized to non-polarized cells around the tumor-like
spheres suggest varying ability for different escape cells.
But the essential is that change in the cell polarity syste m
was associated with 3-D cell growth in the present study.
Interestingly, for human genome unstable oral tumors the
divisions were oriented in skewed/perpendicular posi-
tions relative to the cytoskeleton axis [37], and the ques-
tion is: did this happening involve the present sequence
of genome damage, cycling endopolyploidy with per-
pendicularity, cell senescence, and 3-D escape cell
growth with change in the cell polarity axis?
The acquired growth characteristic of 3-D which is
only known for cancer cells and was presently derived
from normal diploid cells is a new finding. That these
tumor-like spheres grew surface attached in normal liquid

Normal Human Cell Conversion to 3-D Cancer-Like Growth: Genome Damage, Endopolyploidy, 185
Senecence Escape, and Cell Polarity Change/Loss
Figure 2. Fibrblast behavior in the near-senescent phase following exposure to glutamine deficient medium. (a): normal fi-
broblasti array of highly polarized cells with a senescent cell focus in the center (TAS-only). (b): similar cells (TAS-treated)
exposed to glutamine deficient medium. (c): a TAS-only 3-D focus of slightly polarity changed fibroblaststs. (d): mitoses in
cell sheet from TAS-treated cells recovering in complete medium. (e): karyoplast bud-offs from TAS-treated senescent cells.
(f): Tas-treated senescent amorphous cells containing large amounts of lysosomal bodies, insert shows an extrusion process of
these bodies into the medium. (g): small attached escape cells from young senescence. (h): a pre-senescent growth focus with
high mitotic activity and moderate cell polarity change (arrow). (i): Endopolyploid cell division into four equal sized nuclear
products. All illustrations for this 8 × 11 sized plate were enlarged 330 × (including Figure 3). The scale bar represents 25 μm.

Normal Human Cell Conversion to 3-D Cancer-Like Growth: Genome Damage, Endopolyploidy,
186 Senecence Escape, and Cell Polarity Change/Loss
Figure 3. Escape cell-growths from old, deep senescent cells. (a): a small focus of morphologically changed cells to
tri-quadrangular shapes surrounded by flat cells. (b): larger areas showing similar changes with cells on top of each other.
(c)(e): two three dimensional (3-D) cell spheres packed with cells (tumor-like) surrounded by cells with variously changed
fibroblastic polarity, (c): an included cotton tread in the growth sphere. (d): a free-floating clump of cells in the medium.
Scale bar 25 μm for all pictures.

Normal Human Cell Conversion to 3-D Cancer-Like Growth: Genome Damage, Endopolyploidy,
Senecence Escape, and Cell Polarity Change/Loss
187
growth medium is extraordinary. The questions of how
and why this cell growth occurred in deep senescent cul-
tures may as already mentioned hinge on observations by
Chen and Ozanne [19]. Their experiments showed that
deep senescent cells are “active” beyond physiological
processes for survival also indicated earlier [38,39]. The
speculation was that dysfunctional telomeres that caused
TAS can be repaired (gene-conversion recombination?)
during the protracted deep senescent arrest-period [19,
40]. This notion implies occurrence of DNA syn thesis in
senescent flat cells which was shown to occur by a slow,
low rate of H3thymidine nuclear incorporation during a
weeks-exposure time to low H3thymidine concentrations
[38,41,42]. Synthesis of DNA in senescence is highly
controversial [4,9]. Nevertheless, slow repair-synthesis
might in general undo senescence arrest-associated ge-
nomic damage (TAS-treated and ACS) leading to prefer-
ential cell proliferation chance for escape cells from old
as compared to young senescent cells. In the old senes-
cent cultures a karyoplast bud-off process with attach-
ment to free cultural space (Figures 2(e) and (g)) has not
yet been observed, but nuclear isolation for cell escape
can also be achieved by senescence cell-associated cyto-
plasmic compartmentalization [43]. Clearly, this matter,
and the difference in growth capacity for early versus late
senescent escape cells with possible linkage to DNA-
repair synthesis need further clarifications. Similarly, the
3-D growth from TAS-treated and not from TAS-only
escape cells is troublesome because, it points to amino
acid insufficiency being mutagenic by induction of
growth pr omoting m ut a t ions.
But interestingly, the present escape cells have also
gone through the amorphous flat cell stage which does
not express any known type of cell polarity orientations
[44]. Their spread-out shapes (Figure 2(e) and (f)) make
it difficult to see how escape cells can regain a functional,
stabilized, polarity system for growth control by inter-
cell-specific contact points (e.g., tight junctions).Thus,
both ACS and TAS in pre-neoplasia may actually be
promoting phases for augmentation (additional too that
from perpendicularity in endopolyploid divisions) of cell
polarity change with consequence of increased “inde-
pendent existence” [27]. A speculation is that such gai-
ned cell polarity change for escape cells which prior to
senescence demonstrated mother-cell phenotypic sem-
blance, can lead to a discontinuous step/gap in the cellu-
lar, architectural, de-differentiation process. Possible
examples are changes of adenoma to full-blown carci-
noma and nevi to metastatic melanoma [45,46]. Another
specific example that of t(14;18)-associated follicular
lymphoma which following therape-induced repeated
senescence programs, eventually produced escape cells
(?) that showed: “histological “upgrade” of aggressive-
ness to a so-called “transformed” lymphoma type” [4].
Cell polarity change/loss as a modifier of tumor cell-
robustness is consistently not considered in the well-
crafted reviews/reports on molecular-ways for improve-
ments in cancer therapy. Therefore, the above suggested
scenario with involvement of cell polarity change does
not speak well for the possibility of cancer therapy by
induced senescence [1,4] unless the “late” senescent es-
cape route is known and can be controlled.
5. Conclusions
The present study followed normal, diploid cells on a
pathway of cellular events which was one way to tumor-
like 3-D cell growth from morphologically ch anged cells.
In fact there are no other routes known in spite of over
100 years search for a beginning and progression to tu-
morigenesis. The key issues for the present cellular de-
velopments involved: sufficient (?) genomic damage,
endopolyploid perpendicular divisions to cell polarity
change, disruption of contact inhibition, a protracted se-
nescent cell-arrest period, escape cells from senescence,
and growth of escape cells to multilayered tumor-like
spheres. This is a rather gross sequence of events which
however, include several tumorigenesis-associated stages
as for instance, hyperplasia before telomerase activation,
senescence occurrence in pre-neoplasia, with cell escape
into different cancer-cell phenotypes. These similarities
suggest a model role for the present observations in ex-
plorations of initiation and progression in cancer devel-
opment.
6. Acknowledgements
I am very grateful for laboratory space, use of equipment
and encouragements from Drs. David Schnurr and Sha-
ron Messenger and for computer assistance from Mr.
Chao Pan, all of the California Department of Public
Health, VRDL unit.
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